An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays

Signal amplification strategies are widely used for improving the sensitivity of lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized peroxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanoparticles (AuNPs), is used as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips. The assay scheme uses AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG as a detection antibody (dAb), for the detection of human-IgG, as a model analyte. Recognition of the analyte by the capture and detection antibodies is first evidenced by the appearance of a red color in the test line (TL), due to the accumulation of AuNPs. Subsequent addition of 3,3',5,5'-tetramethylbenzidine (TMB) induces an increase of the test line color, due to the TMB being converted into an insoluble colored product, catalyzed by FeMC6*a. This work shows that FeMC6*a acts as an efficient catalyst in paper, increasing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. Furthermore, FeMC6*a achieves lower limits of detection that are found in control experiments where it is replaced with horseradish peroxidase (HRP), its natural counterpart. This study represents a significant proof-of-concept for the development of more sensitive LFiAs, for different analytes, based on properly designed artificial metalloenzymes.