Development of a Magnetic Bead-Based Electrochemical Immunoassay for Human Immunodeficiency Virus Type 1 p24 Antigen Detection

Early and sensitive detection of human immunodeficiency virus 1 (HIV-1) p24 antigen is crucial for identifying acute infection before seroconversion and monitoring low-level viremia. Here, we report a magnetic bead (MB)-based electrochemical immunoassay using stencil-printed carbon electrodes, systematically comparing streptavidin-, tosyl-, and N-hydroxysuccinimide-MBs for p24 detection. Tosyl-MBs achieved superior analytical performance with limits of detection of 19.4 pg/mL in tris-buffered saline with Tween 20, 24.3 pg/mL in media containing viral lysis buffer, and 8.8 pg/mL in human serum, demonstrating excellent matrix tolerance and reproducibility across the clinically relevant concentration range. The sandwich immunoassay employed optimized capture and horseradish peroxidase-conjugated detection antibodies with chronoamperometric readout using 3,3',5,5'-tetramethylbenzidine substrate at 0.0 V vs a silver pseudoreference electrode, enabling rapid immunocomplex quantification on magnet-retained beads with minimal sample handling. Tosyl-MBs provided the strongest concentration-dependent response across all matrices tested, with surface chemistry influencing antibody immobilization efficiency, nonspecific background signal, and p24 capture capacity. The platform demonstrated stability for at least 28 days under storage conditions and successfully distinguished inactivated HIV-1 samples from negative controls, thereby establishing tosyl-MB electrochemical immunoassays that are compatible with serum and other complex biological matrices. Further validation with clinical specimens, integration of simplified sample-processing steps, and implementation in portable instrumentation will be required to assess their potential for future point-of-care HIV diagnostics.