Selection of nitrocellulose membrane (NC)

How to continuously and firmly bind the capture molecules to the detection line T and the quality control line C is the key to producing a sensitive and reliable immunochromatographic test reagent. At the same time, POCT manufacturers can also confirm its performance (such as sensitivity, specificity, etc.) in combination with their own production process, so as to select an NC membrane that meets the actual production needs.

Usually, the following aspects are used to select a suitable membrane during the research and development process:

● Thickness: It can be measured with a thickness gauge, and several points on the NC membrane are selected for measurement to examine its uniformity. If the thickness is uneven, the width of the C/T line drawn will be different. When the amount of capture reagent remains unchanged, the thicker the membrane, the narrower the C/T line, and vice versa.

● Width: It can be measured with an ordinary ruler or a vernier caliper. The commonly used widths are 20mm and 25mm. Manufacturers can customize special specifications according to the needs of the actual production process.
● Backing: NC membranes are available in two types: without backing and with backing. Due to the characteristics of the material itself, NC membranes without backing are very brittle and easy to break. The presence of the backing not only improves the strength of the NC membrane, but also facilitates processing operations during the production process; at the same time, after it is pasted to the PVC rubber sheet, the backing can effectively prevent the migration of substances in the rubber into the NC membrane to avoid affecting the performance.
● Climbing speed (membrane pore size): At present, the industry uniformly adopts the climbing speed test method to characterize the membrane pore size in the lateral flow direction. The climbing speed can be measured by a stopwatch to measure the time (s) taken by purified water to "climb" from the bottom of the membrane to the top of 4cm through capillary action. According to the different climbing speeds, NC membranes can generally be divided into three categories: fast flow rate, medium flow rate and slow flow rate. The faster the flow rate, the larger the pore size.
● Protein adsorption: It is generally recognized that the binding of NC membranes and proteins is the result of the combined action of hydrophobic forces, hydrogen bonds and electrostatic attraction between each other. The strength of the protein-membrane binding ability will directly affect the detection sensitivity and the width of the C/T line.

Generally speaking, factors that affect the binding between NC membrane and protein include:

1) The properties of the membrane material itself: Although NC membranes produced by different manufacturers are all made of nitrocellulose, there are certain differences in the performance of different raw materials, which will lead to different binding abilities with proteins.

2) Specific surface area: The larger the specific surface area, the larger the surface area of the polymer in the membrane, the more binding sites that can be used with proteins, and the stronger the protein binding ability.

3) The content of surfactant: Surfactants play a wetting role on NC membranes, but too high a concentration may affect the binding of NC membranes with proteins.

Optimization of antibody pairs
Although the physical composition, construction technology and buffer of the test strip play a major role in optimizing the test, the selection of antibody pairs is still the core of the entire process and needs to be carefully screened to ensure the affinity, specificity and stability of the antibodies. Monoclonal antibodies (mostly derived from mouse hybridomas) are widely sought after by scientists in the development of in vitro diagnostic reagents due to their high yield and good stability.

Selection of nanoparticles

When selecting nanoparticles, the first thing to do is to determine the target of the developed diagnostic reagent, qualitative or quantitative detection, multi-indicator detection or single indicator detection, so as to finally determine the use of colloidal gold, latex microspheres or quantum dots, fluorescent microspheres and other materials. Of course, in addition to this, it is also necessary to consider whether the selected material has the following characteristics:

● Colloidal stability in solution under various conditions and temperatures;
● Detection sensitivity within a large (and useful) dynamic range;
● Efficiency and reproducibility of conjugation (without loss of chemical and biological integrity and activity);
● Lack or very low nonspecific binding properties (ensuring high signal-to-noise ratio);
● Appropriate cost to achieve commercialization;