A Portable Personal Glucometer-Based Immunoassay Platform for Horseradish Peroxidase-Related Biomarker Detection

Given the prevalent use of horseradish peroxidase (HRP) as an enzyme label in immunoassays, the practical importance of point-of-care testing for the portable identification of HRP-related biomarkers is evident. Accordingly, this study devised a portable immunoassay platform using a personal glucometer (PGM) to achieve rapid detection of HRP-related biomarkers. 3,3′,5,5′-tetramethylbenzidine (TMB) and dopamine (DA) were used as signal-regulating substrates, and the assay principle was based on the HRP-mediated redox reaction. In the absence of HRP, TMB and DA directly reacted with [Fe(CN)6]3− on commercial glucose test strips, producing a strong PGM response. When HRP and H2O2 were present, HRP facilitated the oxidation of TMB to generate oxidized TMB, which further reacted with DA to produce oxidized products with weak reactivity toward [Fe(CN)6]3−, resulting in a decreased PGM signal. Conducting the quantitative assay involved measuring the HRP-dependent change in the PGM readout. Unlike many PGM immunoassays that rely on additional glucose-producing enzyme labels or amplified nanomaterial tags, the proposed method directly converts the signal of the conventional HRP label into a PGM-readable response. The sensing platform demonstrated a good linear response to HRP concentrations ranging from 156 to 625 ng/mL, with a low detection limit of 26 ng/mL. Interference experiments indicated good tolerance to the tested nonspecific proteins. The preliminary applicability of the platform was further evaluated by detecting cardiac troponin I (cTnI) and heparin-binding protein (HBP) in spiked serum samples, with recoveries ranging from 93.0% to 117.1% for cTnI and from 88.3% to 112.5% for HBP. In summary, the proposed platform provides a portable digital readout for HRP-linked immunoassays and reduces the dependence on dedicated readout instruments.