Electrophoresis‐Assisted Multilayer Assembly of Nanoparticles for Sensitive Lateral Flow Immunoassay**

Abstract Lateral flow immunoassay (LFIA) is a rapid, simple, and inexpensive point‐of‐need method. 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 by electrophoresis. The nanoparticles link to immunocomplexes 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 −1 , making it clinically acceptable. Testing 118 clinical samples for hepatitis B showed that signal enhancement increased the diagnostic sensitivity of LFIA from 73 % to 98 % while not affecting its 95 % specificity. Electrophoresis‐driven enhancement of LFIA is universal (antigen‐independent), takes two minutes, and can be performed by an untrained person.