OXIDATIVE DYES-INSPIRED PEROXIDASE SUBSTRATES SIGNIFICANTLY IMPROVE THE SENSITIVITY OF ENZYMATIC LATERAL FLOW IMMUNOASSAYS

Lateral Flow Immunoassays (LFIAs or LFAs) have emerged as indispensable paper-microfluidic tools for point-of-care medical diagnostics.However, these assays exhibit inferior sensitivities compared to laboratory techniques like the enzyme-linked immunosorbent assay (ELISA), and hence, enhancing their sensitivity remains a significant challenge [1].This work presents a strategy to address this issue by introducing novel oxidative coupling-based chromogenic substrates of horseradish peroxidase (HRP) for enzymatic signal amplification in LFAs.The development of these new enzymatic substrates is inspired by the chemistry of oxidative permanent hair dyes which consist of two components: a primary intermediate and a coupler.These undergo an oxidative coupling electrophilic substitution reaction in the presence of H2O2 and HRP as a catalyst to produce an intensely colored dye.These novel substrates have enabled our LFIAs to outperform those employing conventional substrates, such as 3,3'-diaminobenzidine (DAB) and 3,3',5,5'-tetramethylbenzidine (TMB), achieving an unprecedented sub-ng/mL limit of detection (LoD) for Human IgG as a model analyte.Specifically, our LFIAs can detect Human IgG at 0.16 ng/mL (1.07 pM), which is an order of magnitude better than the best LoD (1.2 ng/mL) reported in the literature [2].Furthermore, LFIAs utilizing these substrates exhibit over 40 times improved sensitivity than the conventional gold nanoparticle-based LFIAs and are at least two orders of magnitude cheaper compared to conventional substrates, making them an attractive choice for point-of-care diagnostics in low-resource settings.