Once the conjugate is optimized, the next step is to dry the conjugate onto a conjugate pad. The conjugate pad is essential to maintaining the integrity of the conjugate upon drying, aiding in the long-term stability of the conjugate, and ensuring that the conjugate completely and uniformly releases after wetting with the sample media or buffer. Thus, the selection of the appropriate conjugate pad, as well as developing the right conjugate pad treatment plan, is essential for any lateral flow assay.
Conjugate Pad Selection & Treatment
There are many conjugate pads commercially available from multiple suppliers, such as Millipore and Ahlstrom. A conjugate pad should provide:
- Consistent and uniform conjugate release
- Low non-specific binding of the conjugate or sample
- Consistent uptake of conjugate when dried down
Typically, the material in which conjugate pads are made are either glass fiber or cellulose filter. Cellulose filters typically have higher volume retention than glass fiber. Within these two groups of material are a variety of options with different densities (g/m2) and thickness (µm). These two factors contribute to what is called the “bed volume” (i.e. total volume of air in the material). Bed volume is critical in conjugate uptake and release. For example, Ahlstrom 8950 and Millipore GFDX are both glass fiber conjugate pads that perform very differently due to differences in bed volume. Ahlstrom 8950 is a relatively low-density glass fiber that will release the sample and/or conjugate onto the membrane quickly. A fast release rate is often beneficial for competitive assays and for viscous sample mediums, such as saliva. Millipore GFDX however, is more dense material that can hold a larger volume of conjugate, and release the conjugate slowly. Most decisions on material will be based on specific assay needs such as speed, and conjugate concentration. It is best to try an array of material to best understand how your assay is impacted by the material and physical properties of the conjugate pad.
Conjugate Pad Treatment
Once a conjugate pad has been selected, it may be necessary to pre-treat the conjugate pads before dispensing conjugate. The pre-treatment components often include a salt buffer to account for differences in sample pH, as well proteins, polymers, and detergents that can aid in release of the conjugate from the conjugate pad. A consistent and uniform release is essential for all lateral flow assays. Components of the conjugate pad treatment buffer will also move up the strip faster than the conjugate, and can help block protein binding sites on the membrane prior to the conjugate interaction. This will lead to less non-specific binding, and higher sensitivity. When optimizing the conjugate treatment, keep in mind that too much salt or other reagents can have negative effects. A high concentration of salt can cause the nanoparticles to aggregate, and too much protein or detergent can block the conjugate from adhering to the material. The formulation of the blocking buffer will need to be optimized for each specific lateral flow assay. Generally, adding blocking reagents and non-ionic surfactants to the conjugate can eliminate the need for blocking the nitrocellulose membrane. Conjugate pad treatment can be performed by immersion (i.e. dipping the cut conjugate pad in a pool of blocking solution, or by spraying uniformly with an automated dispenser. At nanoComposix we typically dry our conjugate pad treatments in a forced air convection oven at 37°C for 1-2 hours, and then cure and store in a desiccated environment (<20% relative humidity) at 18-25°C.
Drying Conjugate Onto Conjugate Pad
Conjugate is typically applied to the conjugate pad with the use of an air jet dispensing platform or by immersion. Several machines with hollow fiber dispensers used to stripe nitrocellulose membranes can also be configured with an air jet spray apparatus to dispense the conjugate onto the conjugate pads (e.g. Isoflow, BioDot, and Kinematic). The use of a dispensing platform allows for a controlled and consistent quantity of conjugate to be loaded into each pad. When developing a semi-quantitative or quantitative assay, a very high consistency between strips is needed in order to reach the required precision. The immersion technique is traditionally more variable in the amount of conjugate loaded into each pad, and only recommend if a dispensing platform is not available.
The buffer used to apply the conjugate onto the conjugate pad is also important for the success of your assay. Typically a low concentration buffer, such as 5mM Borate, with the addition of a stabilizing agent (i.e. Sucrose) is used to ensure long-term stability of the dried down conjugate and re-solubilization once it interacts with the sample. A recommended starting concentration of sugars for gold conjugates between 10-20 OD is 10% sucrose and 5% trehalose, although this should also be optimized to improve, flow, stability, and test results.
A typical starting dispense rate is 10 µL/cm of 40 nm Au at OD 10, or 15 µL/cm of 150 nm gold nanoshells at 20 OD. Although this is a starting point, the optimal dispense rate and OD can vary dramatically depending on the assay. After dispensing the conjugate, we dry our conjugate pads in a forced-air convection oven for 1 hour at 37˚C. The dried conjugate pads are then cured overnight in a desiccated environment with <20% humidity prior to testing.