Talk, day 4 October, 12 h, Living room of Degrees of Chemistry.
"Immunoassaig de flux lateral millorat per electroforesi per a l'anàlisi altament sensible d'antígens virals"
Electrophoresis-enhanced lateral flow immuno-assay for highly-sensitive analysis of viral antigen
Sergey N Krylov
Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada
Point-of-need detection methods are required for effective medical testing, food quality control, environmental monitoring, etc. Lateral flow immunoassay (LFIA) is a uniquely simple point-of-need method. It does not require analytical instrumentation and can be performed rapidly by non-trained individuals in resource-limited settings. LFIA utilizes test-strips: preassembled overlapping membranes loaded with dried immunoreagents. A drop of the liquid sample is placed on the strip and capillary forces move it laterally through the membranes. Sample’s moving through the zones of dried immunoreagents causes the rehydration of the latter and facilitates affinity binding of the labeled antibodies to the analyte. This binding leads to the formation of a zone with labeled immunocomplexes on the strip. Nanoparticles of different nature are typically used as labels to facilitate visual (colorimetric) detection of the immunocomplexes. A qualitative result is obtained within 15 min by visual examination of the test strip for the presence and/or absence of two (or more) colored zones. The rapidness, simplicity, and low cost of LFIA makes it an undisputed preference for mass-screening of population during pandemics. It is also a method of choice for home tests, such as a classic pregnancy test. On the downside, the limit of detection (LOD) of LFIA is too high for it to be applicable to samples with low concentrations of the analyte, which makes it impossible to use LFIA for the detection of low-abundant disease biomarkers and pathogens. Reducing the LOD of LFIA is an important task which attracts a great and growing attention of the research community. Often, the improvement of LOD is achieved via compromising one or more of the three key benefits of LFIA.
To overcome limitations of LFIA, we introduce a signal-enhancement procedure that is performed after completing LFIA and involves controllably moving biotin- and streptavidin-functionalized gold nanoparticles along the test strip by electrophoresis. The nanoparticles link to immunocomplexes and each other forming multilayer aggregates on the test strip, thus, enhancing the signal. Here, we demonstrate lowering the LOD of hepatitis B surface antigen from approximately 8 to 0.12 ng/mL, making it clinically acceptable. Testing 76 clinical samples of serum and plasma for hepatitis B revealed that signal enhancement increased diagnostic sensitivity of LFIA from 72% to 98% while not affecting its 90% specificity. Electrophoresis-driven detection enhancement of LFIA is universal (antigen-independent), takes two minutes, and can be performed by an untrained person using an inexpensive accessory.
