Novel 6xHis/HaloTag mammalian expressed autoantigens for the detection of humoral response with multiplexed electrochemical biosensors: A breakthrough in colorectal cancer and Alzheimer's disease personalized diagnostics

The integration of autoantibody detection with electrochemical biosensors allows the development of a novel strategy for managing colorectal cancer (CRC) and Alzheimer's disease (AD). This work reports the use, for the first time, of sustainable receptors, autoantigens expressed in mammalian cells, fused to 6xHis at the N-terminus and HaloTag at the C-terminus, immobilized on chloroalkane-modified magnetic beads (MBs) to selectively capture plasma autoantibodies. Detection was based on amperometric measurements using disposable multiplexed screen-printed carbon electrodes (x8 sensing surfaces), horseradish peroxidase (HRP)-conjugated secondary antibodies, and the HQ/H₂O₂ system. HaloTag immobilization gave rise to a great sensitivity allowing discrimination between patients and healthy individuals in comparison with HisTag or -COOH immobilization. The CRC biosensor integrated seven CRC-specific full-length autoantigens, while the AD bioplatform combined three AD-specific full-length proteins and four AD-specific peptides, which allowed a robust diagnostic performance validated by ROC analyses. These multiplexed biosensors provide minimally invasive, cost-effective, and sustainable alternatives to traditional diagnostic methods, and show ideal for discovery of new targets and for mass screening and early disease detection, supporting personalized medicine approaches.