Signal enhancement strategy for a micro-arrayed polydiacetylene (PDA) immunosensor using enzyme-catalyzed precipitation

Abstract

This paper describes a signal enhancement strategy to improve the sensitivity of an antibody-based immunosensor that uses polydiacetylene (PDA) liposomes to detect a target protein (human immunoglobulin E [hIgE]). To achieve ultrasensitive detection, multiple stimuli applied to PDA immunosensor chips offer a signal enhancement method that combines the primary immune reaction between antigen and antibody with the sandwich method of polyclonal antibody (pAb)-conjugated horseradish peroxidase (HRP). In the second step, fluorescence is enhanced by the mechanical pressure from the precipitate formed by enzyme catalysis. In order to detect hIgE, the surface of immobilized PDA liposomes was conjugated with monoclonal antibodies against hIgE, and fluorescence signals were detected after the antigen–antibody reaction. In this step, hIgE concentrations as low as 10ng/mL were detected. Fluorescence signals slightly increased when anti-hIgE pAb-HRP was used as an amplifying agent after primary immunoresponse. After secondary immunoresponse, HRP-catalyzed oxidation of 3,3′-diaminobenzidine produced an insoluble precipitate that strongly stimulated PDA liposomes by their weight and pressure, thereby dramatically increasing the fluorescence signal. Thus, PDA liposome immunosensor could detect hIgE concentrations as low as 0.01ng/mL, representing a 1000-fold increase in sensitivity over the signal generated by the primary immunoresponse. This study indicates that increasing the external mechanical force applied to PDA liposomes by enzyme-catalyzed precipitate formation enhanced the sensitivity of the PDA liposome immunosensor chip. This strategy can be applied to the detection of other biomolecules in experimental or clinical settings where ultrasensitive and highly specific biosensing is required.