Part 2: Membranes in Point-of-Care Diagnostics: Applications and Technologies

Last month, we published the first installment of this 2-part series wherein we discussed lateral flow immunoassay.

Flow-through Immunoassay
Flow-through assay (FTA) is a diagnostic test that allows users to detect the presence of specific biomarkers, typically antibodies, in a sample such as blood. Flow-through assays are particularly useful in POC settings, allowing rapid detection of specific biomarkers without specialized lab equipment and extensive training. They aid in diagnosis and enable quicker therapeutic action [1].

Flow-through Immunoassay relies on the same basic principles as LFIA format with some modification. The most apparent difference between these two methods being the vertical and lateral flow of fluid. The Flow-through Immunoassay can be used for rapid detection of antigens in the sample of interest, which can come from different application areas such as clinical, veterinary, agricultural, food, biodefense and environmental industries [2]. 

Flow-through tests typically come in the form of cassettes divided into four parts: an upper casing, a reactive membrane panel, an absorbent panel, and a lower casing. To perform a test, a diluted sample is applied to the reactive membrane panel and flows through to the absorbent pad, with the target analyte being captured in the membrane. The membrane is then washed to remove unbound, non-target molecules, washed again with a solution containing a signal reagent, and washed again to remove unbound signal reagent. If the analyte was present in the original sample, then by the end of this process it should be bound to the membrane, with the signal reagent bound to it, revealing (usually visually) the presence of the analyte on the membrane

Figure 3 shows the cartridge configuration for Flow Through Immunoassay

 

In FTA the liquid passes vertically through the reaction membrane, so the filtration properties of the membrane are more important than capillary rise times. In principle, all membrane types that have a high and consistent protein binding could be used. However, for the same reasons as for lateral flow immunoassays — wettability, easy capture protein, and reliability — nitrocellulose is again the material which is most frequently used. 

For diagnostic devices where sensitivity and reproducibility are important, track-etched membrane can serve as a critical functional component. The track-etched membranes, such as PETE and PCTE, show excellent results when filtering out certain analytes of saliva, blood, urine, or other targeted fluids and can remove unwanted substances from a test stream.

Dipstick Colorimetric Assays
Dipstick assays are simple and cost-effective POC tests [3]. The test relies on the vertical flow of liquid samples through a porous membrane impregnated with dried reagent pads that interact with the analyte on exposure. Dipstick colorimetric assays are commonly used for various applications, including detecting glucose, protein, pH levels, and specific antigens. These assays provide a rapid test that is easy to read, making them valuable for POC diagnostics. 

Figure 4 shows the example of Dipstick for Colorimetric assays

 

Dipstick test pads require membranes with high wettability, mechanical stability, consistent absorption properties, and rapid flow characteristics. The most important consideration for dipstick test pads is absorption capacity. Therefore, cellulose based membranes are usually recommended for this type of assay because they exhibit excellent absorption capacity, which allows the pad to handle a large sample volume without causing the test strip to fail or decompose from wet mechanical stress. The main components of the Dipstick configuration are:

  • Cellulose Pad: The dipstick contains a cellulose pad that serves as the detection zone. This pad is impregnated with specific detection reagents that give a color reaction when processed. The color change indicates the presence of the target analyte in the sample.
  • Detection Reagents: These reagents are specific to the analyte being tested. They react with the analyte, leading to a visible color change. For example, if the dipstick is used to detect glucose, the reagents would react with glucose molecules in the sample.
  • Sample Application Area: This is where the liquid sample (e.g. urine, blood, or other bodily fluids) is applied. The sample flows through the cellulose pad, carrying the analyte with it. The cellulose pad acts as a flow channel, allowing the sample to move vertically through the dipstick.
  • Color Readout: At the end of the dipstick, there is a designated area where the color change becomes visible. Users can read the result by comparing the color intensity or pattern to a reference scale. Some dipsticks include a control line. The control line ensures that the test is functioning properly. It appears regardless of the presence of the analyte. If the control line does not appear, the test may be invalid. 

Molecular Assays

Molecular assays, also known as molecular diagnostic tests, involve the analysis of genetic material (DNA or PNA) to detect specific sequences, mutations, or variations [4]. These tests provide valuable information for disease diagnostics. In recent years, several molecular POC assays have been developed including steps of nucleic acid isolation and cell isolation. These tests require membranes to facilitate efficient nucleic acid isolation, ensuring high-quality samples for downstream applications like PCR, sequencing, and disease diagnosis. The choice of membrane is critical for molecular POC assays and selection of the right membranes ensures optimal performance, accuracy, and efficiency in POC tests.

Glass fiber membranes are especially popular for nucleic acid isolation. They offer high nucleic acid binding capacity and high wet strength compared to other membranes. The optimal membrane will depend on several characteristics such as the required thickness, pore size, porosity, and surface quality. Glass fiber filters are cost-effective materials for developing molecular diagnostics and nucleic acid kits for nucleic acid extraction.

Cell isolation steps require the membrane with highly defined pore sizes to effectively isolate the specific target cells. The most suitable option is track-etched membranes. These membranes are manufactured using a precise etching process to create uniform and cylindrical pores, resulting in well-defined porous structure for accurate particle separation. Track-etched membranes allow selective passage of particles or cells based on their size, making them useful for precise size-based separation. Also, the transparent nature of track-etched membranes enables visualization and examination of filtered samples. 

 

References:

1. St John A, Price CP (August 2014). "Existing and Emerging Technologies for Point-of-Care Testing". The Clinical Biochemist. Reviews / Australian Association of Clinical Biochemists. 35 (3): 155–67. PMC 4204237. PMID 25336761.

2. Valkirs GE, Barton R (September 1985). "ImmunoConcentration — a new format for solid-phase immunoassays". Clinical Chemistry. 31 (9): 1427–31. doi:10.1093/clinchem/31.9.1427. PMID 4028392

3. Colorimetric protein assay techniques - PubMed (nih.gov)

4. Burtis CA, Ashwood ER, Bruns DE (2012). Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. Elsevier. ISBN 978-1-4557-5942-2.