Quantification of Glycated Hemoglobin in Total Hemoglobin by a Simultaneous Dual-Signal Acquisition Approach

The glycated hemoglobin (HbA_1c) level, which is defined as the ratio of HbA_1c to total hemoglobin (tHb, including glycated and unglycated hemoglobin), is considered one of the preferred indicators for diabetes monitoring. Generally, assessment of the HbA_1c level requires separate determination of tHb and HbA_1c concentrations after a complex separation step. This undoubtedly increases the cost of the assay, and the loss or degradation of HbA_1c during the separation process results in a decrease in the accuracy of the assay. Therefore, this study explored a dual-signal acquisition method for the one-step simultaneous evaluation of tHb and HbA_1c. Quantification of tHb: graphene adsorbed carbon quantum dots and methylene blue were utilized as the substrate material and linked to the antibody. tHb was captured on the substrate by the antibody. The unique heme group on tHb catalyzed the production of •OH from H₂O₂ to degrade methylene blue on the substrate, and a quantitative relationship between the tHb concentration and the methylene blue oxidation current signal was constructed. Quantification of HbA_1c: complex labels with HbA_1c recognition were made of ZIF-8-ferrocene-gold nanoparticles-mercaptophenylboronic acid. The specific recognition of the boronic acid bond with the unique cis-diol structure of HbA_1c establishes a quantitative relationship between the oxidation current of the label-loaded ferrocene and the concentration of HbA_1c. Thus, the HbA_1c level can be assessed with only one signal readout. The sensor exhibited extensive detection ranges (0.200-600 ng/mL for tHb and 0.100-300 ng/mL for HbA_1c) and low detection limits (4.00 × 10^-3 ng/mL for tHb and 1.03 × 10^-2 ng/mL for HbA_1c).