The Progressive Movement from Qualitative Lateral Flow Assays to Modern Quantitative Lateral Flow Assays

Author: Sean Lightheart

A Lateral Flow Assay (LFA) utilises simple technologies to allow for rapid and low-cost detection of the presence or absence of a target analyte with the results determinable at the point of care. Their applications can range from determining biomarkers in human disease to contaminants in a water supply, but the most commonly used LFA is the rapid pregnancy test (1). LFAs are usually in the form of a strip that contains various components. A liquid sample (e.g., blood, urine, saliva) is deposited on an absorbent pad located at one end of the strip. The liquid is then drawn along the strip, via capillary action, in which it will first encounter the conjugate release pad. This pad contains antibodies, specific to the analyte in question, that have been conjugated to coloured or fluorescent particles (colloidal gold or latex microspheres) (2). The sample now containing the analyte bound to conjugate antibodies continues along the strip to the test line and control lines. The lines contain immobilised antibodies specific to the target analyte (3). Successful binding of the analyte at the test line will present a coloured line on the test and indicated the presence of the analyte in the sample. Whilst the presence of a coloured line at the control section indicates correct flow of sample liquid down the test. Determination of results is qualitative; however, the assays can be combined with a dedicated optical reader to provide quantitative results.

Difficulties with qualitative LFAs

Whilst LFAs are an impressively useful tool that are able to produce rapid results at the point of care, some key attributes have been sacrificed for speed and cost. One of the most substantial disadvantages is the poor limit of detection (LoD) achievable by LFAs. With a high LoD the tests can often produce false negatives as they are unable to detect concentrations of the target analyte on a small enough scale (4). This results in many LFAs being used only as a screening process with more in-depth laboratory tests required for confirmation of results. Additionally, reproducibility between tests is often not achievable, with varying sample volumes and qualitative results determined by human interpretation (5). Finally, with qualitative results that are often determined by the patients themselves, there is no formal digital storage, meaning results are lost when the test is discarded.

Advantages of quantitative electrochemical LFAs

More recently, electrochemical readers have been incorporated into LFAs to take results from being qualitative “yes/no” answers to provide quantitative results. They work in the same manner that a standard LFA would work however, at the normal test line there is an electrochemical sensor, that is able to correlate different signal intensities with different quantities of the target analyte via binding and thus give the more accurate, quantitative results (6).

One such advantage of electrochemical LFAs is the enhanced LoD that they offer. The electrochemical sensors are able to detect much lower concentrations of the target analyte than standard LFAs which allows for a more accurate diagnosis and reduces the likelihood of false results (6).

However, the greatest advantage separating electrochemical LFAs from standard LFAs is the ability to provide quantifiable results. The intensity of the signal that is produced correlates to the concentration of analyte that was bound. This means the tests are not only able to determine if the analyte is present but give more meaningful results via quantification (7). A key example of this is the Clearblue digital pregnancy test, that not only determines if the individual is pregnant but by measuring quantities of the analyte, provides them with information on how far along the pregnancy they are. Furthermore, quantifiable results eliminate human error that could come with qualitative results. The quantitative results are produced digitally. This allows the potential for results to be automatically sent from the point of care, to a health professional elsewhere for a greater interpretation of the results or to be uploaded to a cloud-based storage for access at a later date.

Finally, precise testing such as ELISAs are expensive and require bulky laboratory equipment. Electrochemical technologies have allowed for rapid, accurate testing at the point of care, whilst maintaining low cost and ease of use (8).

Electrochemical LFA areas of development

Although combining electrochemical sensors into LFAs is said to improve many aspects of the assays, there are still certain aspects that could be developed to produce a more complete electrochemical LFA.

The connection between the sensor surface and the test site has to be extremely intimate. Therefore, further development of an extremely thin and flexible sensor, will allow the sensors to maximise the connection with the membrane and enhance the sensitivity of the assay.

Another area of development is the ability of the analyte to diffuse from the nitrocellulose membrane down to the sensor surface. By optimising the nitrocellulose membrane this will facilitate extra diffusion and in turn increase the precision of the assay (9).

If you are looking to develop a low-cost electrochemical lateral flow assay, contact us today.

 Reference:

1. Lateral Flow Assays. Koczula, Katarzyna M and Gallotta, Andrea. 1, s.l. : Essays In Biochemistry, 2016, Vol. 60.

2. Comparative Study of Gold and Carbon Nanoparticles in Nucleic Acid Lateral Flow Assay. Porras, Juan Carlos, et al. 741, s.l. : Nanomaterials, 2021, Vol. 11.

3. Designs, formats and applications of lateral flow assay: A literature review. Sajid, Muhammad, Kawde, Abdel-Nasser and Daud, Muhammad. 6, s.l. : Journal of Saudi Chemcial Society, 2015, Vol. 19.

4. An improved detection limit and working range of lateral flow assays based on a mathematical model. Lui, Zhi, et al. 12, s.l. : The Analyst, 2018, Vol. 143.

5. Merk Millipore. Rapid Lateral Flow Test Strips - Considerations for Product Development. s.l. : EMD Millipore Corporation, Billerica, MA, 2013.

6. Electrochemical Lateral Flow Devices: Towards RapidImmunomagnetic Assays. Ruiz-Vega, Gisela, et al. 10, s.l. : ChemElectroChem, 2017, Vol. 4.

7. Towards Lateral Flow Quantitative Assays: Detection Approaches. Urusov, Alexandr, Zherdev, Anatoly and Dzantiev, Boris. 3, s.l. : Biosensors (basel), 2019, Vol. 9.

8. Biosensors: sense and sensibility. Turner, Anthony P.F. 8 , s.l. : Chemical Society Reviews, 2013.

9. Integrating high-performing electrochemical transducers in lateral flow assay. Perju, Antonia and Wongkaew, Nongnoot. s.l. : Analytical and Bioanalytical Chemistry, 2021.