Palladium nanostructure-based label-free electrochemical biosensors for HbA1c and insulin measurement

Diabetes mellitus (DM) is characterized by impaired glucose regulation due to insufficient insulin secretion or cellular resistance to insulin. Although insulin is the primary regulatory molecule, glucose remains the most monitored biomarker despite its high variability. Glycated hemoglobin (HbA1c), a more stable marker with a longer half-life, provides a comprehensive assessment of long-term glycemic control. Simultaneous measurement of HbA1c and insulin would offer enhanced insight for DM management. In this study, we present a label-free electrochemical biosensor capable of simultaneously quantifying HbA1c and insulin using a single drop of blood. The device integrates dual screen-printed carbon electrodes functionalized with palladium nanostructures (PdNS), allowing covalent immobilization of anti-HbA1c and anti-insulin antibodies on WE1 and WE2, respectively. Upon incubation with a pretreated blood sample and introduction of hydrogen peroxide (H₂O₂) serving as the measuring solution, amperometric signals were generated: oxidation of the heme group in HbA1c at WE1, and suppression of current at WE2 due to insulin interference. All concentrations were quantified based on current responses to the measuring solution. The biosensor demonstrated excellent analytical performance, including high sensitivity (R 2 > 0.96), selectivity (>95 %), long-term stability (>95 % over 120 days), and operational efficiency, as confirmed through standard and whole blood sample analysis. This is the first disposable point-of-care (POC) device that enables dual detection of HbA1c and insulin on a single chip, offering a practical and comprehensive tool for real-time DM monitoring and personalized disease management. A. Schematic illustration of biosensor chip preparation for HbA1c and insulin detection, along with SEM images of bare and PdNS-deposited carbon electrodes. B. Workflow for HbA1c extraction from whole blood prior to biosensor analysis. Representative amperometric curves are shown for different samples: black for blank, red for HbA1c, and blue for insulin. The arrow (red and blue) indicates the change in current with increasing analyte concentration.