DIDICO

Designing a diagnostic device platform

Membrane-based immunodiagnostic devices generally take one of two forms: flow-through devices where the sample flows down through the membrane, concentrating the sample in a small defined area; or, lateral flow devices where the sample is applied at one end of the device and flows laterally through the membrane matrix, binding with secondary and primary antibodies that have been applied on the device matrices during the manufacturing process. In both cases the devices are composed of membrane layered with papers that are used to increase capillarity and control flow rates.

Flow-through assays

In flow-through assays, first developed in the mid-1980s, reactants are drawn through the membrane due to its contact with an absorbent bed. This brings first the analyte and subsequently the tagged secondary antibody into the void volume of the membrane. This configuration dramatically reduces the diffusional time requirements experienced when the reactants in solution must diffuse to the immobilized protein on the surface of the membrane as in a Western blot. This immuno-concentration process brings reactants into the matrix of the membrane where diffusional distances are short, ensuring that the kinetics of binding in the solid phase closely approach the kinetics of binding in solution. Nitrocellulose membranes with pore sizes of 0.2 microns to 8 microns have been used in flow-through assays. This type of assay has the advantage of simplicity both in use and in manufacturing.

Lateral flow immunoassays

Lateral flow immunoassay systems have been developed, which allow for single-step assays that require only sample addition. In these types of assays, sample is added to one end of the device and capillaries through the interstitial space of the materials in the device. While continuing along this flow path, the sample contacts dried reagents usually in the form of a tagged secondary antibody, which then migrate with the analyte to a capture zone of membrane-immobilized antibody. Unreacted tagged antibody continues to capillary past this capture zone, normally to an end-of-assay indicator. Generally there is absorbent material at the distal end of these devices to aid in drawing the sample through the device. Another format of a diagnostic test is the dipstick test. A dipstick test uses a membrane-based detection system as in the lateral flow format mentioned above. But the volume is not as well controlled, because the strip is used as a midstream device or is placed directly into a container with excess sample that the device will wick up.

Nitrocellulose membranes — the core of success

While a variety of microporous polymer membranes are available, most current immunoassays use nitrocellulose. This polymer binds most proteins, in particular antibodies, with sufficient density and avidity. NC membrane does not require covalent attachment of the immobilized reactant, in most cases. Additionally, nitrocellulose membranes are the proven membrane of choice because they avoid the high level of nonspecific interactions (background noise) so common with assays run on other polymers. We manufacture NC membranes in a wide range of porosities from 0.05 to 12 microns, offering a choice of binding capacities and wicking or capillary rise rates to aid in the flow of reactant through the assay. The choice of a membrane for an immunodiagnostic device depends on several factors, such as the sensitivity of the assay, the time required to run the assay and type of readout employed in the assay. The pore size of the membrane has an impact on these parameters. The larger the pore size, the lower the surface area and, therefore, the lower the protein binding capacity. However, larger-pore-size membranes support more rapid filtration and lateral flow rates and provide the potential for a faster assay. A smaller-pore membrane has a higher surface area that will yield a higher density of immobilized antibody. Sometimes this can create a more sensitive assay. The size of the tagged antibody complex may also dictate the requirement for the pore size. Assays that use an antibody coupled to a colored latex bead require a larger-pore-size membrane.

Properties

There are several physical properties associated with the NC polymeric surface that influence the way in which a point-of care (POC) test performs. Below, we describe the five main properties and how they affect lateral flow test performance. The typical immunodiagnostic assay for POC analysis is based on a device using a large-pore-size membrane. This kind of device takes advantage of the lateral flow of fluids through a membrane, enabling the application of various immunoreactants at different locations along the membrane strip. Varying the pore size permits control of the rate of flow through the membrane and therefore control of reaction time. 

Porosity

The reactant must flow through the membrane so that it can separate bound from free components. Pore size affects both the lateral flow rate and the available surface area for protein binding. As pore size increases, the time required for a sample to wick a particular distance decreases. Capillary rates decline with increasing distance wicked.

Pore size has an impact on the protein binding capacity and potentially the sensitivity attainable. And pore size is not the only factor to affect capillary rates. Most nitrocellulose membranes contain a surfactant to aid in wetting and act as an antistatic agent. The amount and type of surfactant can be altered to change the capillary rates of membranes with the same pore size. Pore size and surfactant content must, of course, be appropriate for the time requirement of the assay. Capillary rates can be affected by the blocking agent used. A system called “block-on-the-fly” can avoid this phenomenon. This is done by impregnating the sample application pad with a soluble, non-ionic surfactant. When the sample is added to the device, the detergent will dissolve and move with the sample through the membrane, blocking as it moves. For a user, what is most critical is the actual amount of sample delivered to the control and test lines – “the reaction zones.” To ensure that a readable test result is developed at the test site, enough reacting species have to be brought together. Often, a certain residence time in the reaction zone is also needed. Obviously, the more time you give it, the more volume will be delivered. Sample type differences are also clear. Serum flows less easily, and therefore less will be delivered in the same time when compared to water. These results mirror the wicking or lateral flow data. A manufacturer’s wicking data generally correlate, though not directly, to the sample-delivering characteristics of the membrane

Symmetry

As a result of the production process, NC membranes demonstrate a slight asymmetry in their overall structure, which is exhibited as “sidedness”. Each NC membrane has both a belt side and an air side. The surface of the belt side of the membrane is in constant contact with the support during membrane formation. The air side is the opposite surface through which solvents are evaporating during the drying process. The air side is usually coarser than the belt side. Although this asymmetry exists for all of the pore sizes, it is restricted to only the first few µm of either side. As the protein binding depends on the pore size, it can be slightly different between sides. Dispensing results can also differ between sides. When testing membranes, many end users will define which surface is to be used in all future production to maintain consistency in performance.

Protein binding capacity

Since most test developers are depositing proteins onto the solid membrane surface of a POC test, the binding capacity of that membrane is critical. Protein binding is primarily influenced by the pore size, with a general decline in protein binding as pore size increases. Binding capacity is also dependent on other factors, including the type of protein and the way the binding is done. Proteins must bind to the solid phase and must also remain immunoreactive for the test to function properly. A binding capacity that is too high or too low can have a negative impact on immunoreactivity as well as on blocking efficiency. Even the largest-pore-size NC, with binding capacities in the range of 20 to 30 µg/cm2, have overall protein binding capacities that will yield strong, clear test signals.

Strength

Nitrocellulose membranes are the media of choice for diagnostic applications. However, if the membranes are used in high tension reel-to-reel application, many customers request added tensile strength. Many device manufacturers find that AE nitrocellulose has sufficient strength for their automated manufacturing processes. Should you require a stronger membrane, nitrocellulose direct-cast onto a plastic support is an excellent alternative.

Nitrocellulose storage conditions

Nitrocellulose membranes should be stored at 15 to 30 degrees Celsius and 40% to 60% relative humidity prior to processing. Ideally, they should be processed under these conditions as well. Following assembly, it is suggested that devices be packaged in heat-sealed foil pouches with a desiccant. Once devices are sealed into pouches, the above storage conditions are not required; the devices should not be frozen, however.