Novel Oxidative Coupling-Based Chromogenic Substrates for Horseradish Peroxidase-Enhanced Lateral Flow Immunoassays: A Highly Sensitive and Economical Alternative to Conventional Substrates

Lateral flow immunoassays (LFIAs) have become indispensable in point-of-care diagnostics, but improving their sensitivity remains a critical challenge. Here, we present a strategy to enhance LFA sensitivity by introducing novel oxidative coupling-based chromogenic substrates for horseradish peroxidase (HRP) enhancement. Motivated by the need for more sensitive and cost-effective LFAs, we developed substrates inspired by oxidative permanent hair dyes. These substrates consist of two components: a primary intermediate and a coupler that undergo an oxidative coupling reaction in the presence of HRP and H2O2 to generate an intensely coloured dye. Our LFAs employing these substrates outperform conventional substrates (3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenzidine (TMB)) and achieve an unprecedented sub-ng/mL limit of detection (LoD) for Human IgG. LFAs employing these novel substrates could detect Human IgG at 0.2 ng/mL, significantly surpassing reported sensitivities in the literature. Compared to gold nanoparticle based LFAs, our substrates exhibit over 30 times higher sensitivity. Moreover, these substrates offer substantial cost savings, making them a compelling choice for point-of-care diagnostics.