Part 1: Membranes in Point-of-Care Diagnostics: Applications and Technologies

July 15, 2024

Introduction
Membranes are widely used for Point-of Care (POC) diagnostics as filters to facilitate sample transport, analyte separation, reagent immobilization, and flow control for rapid tests to detect analytes from various biological samples (blood, urine, saliva, virology and mouth swabs, etc.) [1].

The rapid diagnostic test is a medical diagnostic tool suitable for preliminary or emergency medical screening to provide quick results especially in situations with limited resources.

Depending on the biological samples, rapid diagnostic tests are used for antibodies and antigen testing for various infections, including HIV and COVID-19, influenza, malaria, streptococcal infections, and various pathogens [2].

The selection of membranes and pads based on the material’s performance, including sensitivity, specificity, and flow control, is crucial for developing POC diagnostic tests. There are two main categories for POC assays: Immunoassays which use antibodies to detect analytes such as antigens, drug molecules, or proteins; and Molecular Assays to detect nucleic acids (DNA or RNA) within the samples.

Some of the membrane based POC immunoassays are Lateral Flow Immunoassays, Flow Through Immunoassays, and Dipstick colorimetric assays. Each method uses a different approach for its antibody-antigen detection and requires a specialized set of membranes and pads.

Lateral Flow Immunoassay
One of the rapid tests in medical diagnostics, both at home and in clinical settings, is Lateral Flow Test (LTF) or Lateral Flow Immunoassay (LFIA). Also known as a lateral flow device, this is a simple device designed to detect the presence of a target substance in a liquid sample without the need for specialized and costly equipment [1].

Figure 1 shows the typical configuration of lateral flow immunoassay.

LFIA relies on the specific binding between an analyte and its corresponding antibody or antigen. Traditionally designed assays are composed of a variety of materials, each has a different property and serve one or more purposes. The parts overlap onto one another and are mounted on a backing card using a pressure-sensitive adhesive. The test strip typically consists of several components:

Sample Pad: The first membrane where the sample (e.g. serum, blood, saliva or urine) is applied acts as sponge and holds an excess of sample fluid. The primary function of the sample pad is to transport the sample from the point of application to the other test components. There are two types of materials that are commonly used as sample pads: cellulose fiber and glass fiber filters.
Conjugate Pad: Contains labeled detector reagents (usually antibodies or antigens) which are conjugated to colloidal gold nanoparticles or colored latex beads. As the sample flows through the conjugate pad, the conjugate is released into the sample. If the target molecule (e.g. specific antibodies) is present in the sample, the conjugate binds to it, and both the sample and conjugate then flow together to the test membrane for further detection. Glass fiber is a suitable choice for conjugate release pads because they do not require pretreatment and have consistent absorbency and wicking rates.
Membrane: Used to bind and detect the target molecule. Usually, the membrane has test and control lines. The test line (T-line) is where specific antibodies or other detection molecules are immobilized. Its primary function is to capture the analyte (the target molecule) present in the sample. If the analyte is present, it binds to the antibodies on the test line, resulting in a visible signal (e.g. a colored line) that confirms the presence of the target. The control line (C-line) serves as an internal quality control to ensure that the LFIA is functioning correctly. It contains antibodies that are not specific to the analyte but instead recognize a different molecule (often an unrelated immunoglobulin or protein). If the control line appears, it validates the test and confirms that the assay components are working as expected. If the control line does not appear, it indicates a failed test due to insufficient sample flow or other issues. Nitrocellulose is widely used for LFIA due to the excellent balance between flow rate and assay sensitivity, providing sufficient protein binding to produce sharp and intense capture lines.
Absorbent Pad: Final test component in LFIA to collect the excess of sample fluid. Without the absorbent pad, the flow would stop once it reaches the membrane. By absorbing excess sample volume, the pad prevents backflow into the membrane to ensure that the sample does not interfere with the test and control lines. A variety of cellulose and glass fiber filters can serve as absorbent pads.

Figure 2 shows the architecture of Lateral Flow Immunoassay

During the LFIA test the liquid analyte sample is applied to the strip and flows spontaneously through the sample pad. Once soaked, the sample flows into the conjugate pad, where the reagents initiate a chemical reaction and labeled antibody conjugate is released. In the conjugate release pad, there are conjugate particles (usually colloidal gold nanoparticles) coated with antibodies specific to the analyte. As the analyte sample and detector antibody flow into the porous membrane, they interact. This marks target particles as they pass through the membrane and continue across to the test and control lines. If the analyte of interest is present, it binds to the labeled detector antibody and gives a visible line, which appears at the test line due to the accumulation of the analyte-conjugate complex. The control line always appears, assuming the test is functioning correctly and indicating that the assay worked properly. The user observes the appearance of lines (or lack thereof) at the test and control lines. The interpretation of the test result is typically straightforward: both test and control lines are visible for positive result; only the control line is visible for negative result, and no lines appear indicating invalid result or test failure. The intensity of the test line can vary, providing semi-quantitative information about the analyte concentration. Some LFIA devices incorporate reader systems for more accurate quantification.

Due to simplicity the LFIA finds applications across various fields, such as healthcare (pregnancy test, infectious disease diagnostics, allergy testing) and disease biomarkers. Other applications include screening for animal diseases, monitoring water quality, and testing food production for harmful contaminants.

References: