Electrophoresis-assisted multilayer assembly of nanoparticles for sensitive lateral flow immunoassay

Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive method for point-of-need analysis. A major limitation of LFIA is a high limit of detection (LOD), which impacts its diagnostic sensitivity. To overcome this limitation, 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.