Development and Performance Enhancement of LFIAs

we have enhanced the sensitivity and limit of detection of lateral flow immunoassay tests, exemplified for the detection of human chorionic gonadotropin (pregnancy) through determination of proper test line position. Test line position affects the flow velocity and concentration profile of analytes at the test line, which itself impacts the reaction rate and thus the colour intensity. The flow velocity was precisely modelled, both analytically and numerically, and the predicted analytes' moving front (lateral) velocity was experimentally verified. Evolution of the velocity magnitude was determined to locate points with proper capillary velocity and accumulated bed-volume. This provides insights into the essence of LFIAs performance for simple and easy to practice improvements. The colour intensity was measured by quantifying the RGB of the resulting test line colours. The response colour intensity was enhanced for at least 20%, up to 40% by proper displacement of the test line position, and the limit of detection was cut by half to 10 IU/L.

One of the ongoing challenges in lateral flow Immunochromatographic assays (LFIA), is lowering the limit of detection and enhancing their signal quality, i.e. the color intensity. There are a number of rather costly and complicated processes for this aim, such as the use of functionalized materials/membranes and additional spectroscopic readout units. Nonetheless, there are simple and easy to practice alternatives, to be uncovered by analyzing the essential parameters of immunological reactions. The color intensity of the test line is a function of analytes flow velocity and their reaction rate. Detection pad width and test line position impact the flow velocity and reaction rate kinetics, examined in this paper for the limit of detection (LOD) and test-line color intensity. Firstly, the impact of width on the LOD was examined for human chorionic gonadotropin (pregnancy biomarker). Test line color intensity was measured using five different widths of the detection pad (trapezoidal) and four different test line positions, and the trends observed were explained according to the measured evolution of the velocity along the chromatography paper. With a constant width absorbent pad, LOD was cut by half to 5 mIU/ml by using a narrowing width detection pad, which keeps the wicking velocity higher than normal strips, and compared to them, color intensity increase between 55 and 150%, depending on the concentration. Nevertheless, a widening detection pad might cut the color intensity up to 150%, compared to normal strips, due to a profound decline of the analyte to ligand ratio at the test line. In addition, adequately sending the test line away from the conjugate pad yields the highest possible color intensity, for up to 400% of increase, in lower concentrations and narrowing test pads. However, further distancing the test line downfalls the color intensity.