Development of Multi-HRP-Conjugated Branched PEI/Antibody-Functionalized Gold Nanoparticles for Ultra-Sensitive ELISA

The enzyme-linked immunosorbent assay (ELISA) is the most widely used technique for the selective detection of various analytes due to its advantages of sensitivity, simplicity, versatility, and high throughput. However, conventional ELISA is not sufficient to detect biomarkers at lower concentration ranges, such as low pM levels. Therefore, we developed multi-horseradish peroxidase (HRP)-conjugated branched polyethyleneimine (PEI)/antibody-functionalized gold nanoparticles (mHRP/bPEI/AuNPs) that immobilize a large number of HRP enzymes to lower the threshold for target antigen detection. Briefly, mHRP/bPEI/AuNPs were fabricated by attaching branched PEI with many enzyme molecules to the surface of streptavidin-HRP-coated AuNPs. The fabricated mHRP/bPEI/AuNPs were applied as a detection probe in ELISA, enabling the quantitative detection of the breast cancer biomarker Thioredoxin-1 (Trx-1) in a range from 10 pM to 100 nM and showed 10³ times greater sensitivity than conventional ELISA, with a limit of detection (LOD) of 1.7 pM for Trx-1. These results suggest that the higher number of enzymes present in mHRP/bPEI/AuNPs amplifies the signal and increases the detection sensitivity. Consequently, we expect that mHRP/bPEI/AuNPs can be used in situations requiring the detection of low concentrations of biomarkers, such as early disease diagnosis.