Sample and Absorbent Pad Selection for Lateral Flow Assays
Both the sample pad and the absorbent (wick) pad are critical components to any lateral flow assay. In this section we will cover the characteristics of each component and how it relates to selection, as well as treatment of the material to improve assay performance.
Sample Pad Selection and Treatment
The sample pad serves two critical purposes:
- Allows for the controlled release of sample and conjugate onto the membrane or conjugate pad depending on strip confirmation.
In some sample mediums, such as urine and saliva, the composition of the sample can vary significantly between individuals depending on a variety of factors (e.g. time of day, diet, age, etc.), and these differences in composition and pH, may interfere with the detection of the target analyte.
The sample pad, being the first component of the lateral flow test strip that comes into contact with the sample, provides an opportunity to control for these differences. Sample pad material and pre-treatment should be evaluated to ensure that the sample has the optimal flow characteristics before the sample reaches the other reagents in the system. - Acts as a filter for unwanted components of the sample while allowing the target analyte to pass through unimpeded.
In some sample matrices, such as whole blood and solubilized solids, it is critical to prevent the passage of unwanted material, so for these samples, the sample pad can act as a filter.
The sample pad will retain the unwanted particulates while allowing the fluid containing the analyte of interest to flow through the test strip. In the case of whole blood, a filter can be used that will hold back the red blood cells while allowing the plasma/serum to pass through the assay.
When selecting the correct sample pad for your lateral flow assay, the following attributes should be considered:
Thickness/Weight
A thicker membrane will have a higher bed volume (i.e. volume of air that can be displaced) allowing for a higher volume of sample to be added. However, thicker membranes will also require larger housings as compression of the sample pad will greatly reduce the absorption of the sample into the sample pad.
Tensile Strength
Low tensile strength material can be very difficult to work with, particularly when it comes to the lamination process. This is an important consideration when preparing for manufacturing.
Material
Typical sample pads consist of cellulose fiber filters and/or woven meshes. Woven meshes (e.g. glass fiber) typically have low bed volumes meaning that very little sample is retained, however they have good tensile strength resulting in easy handling.
Due to the characteristic of the mesh to not retain volume, woven meshes are very impractical for pre-treatment as the blocking reagents will not stay within the sample pad. Cellulose fibers filters have higher bed volumes, lower tensile strength, and are excellent for loading with a variety of buffers and blocking reagents.
There are 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.
Absorbent (Wick) Pad Selection
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 run 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 total 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.
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