Hybrid Nanoflower‐enhanced Enzyme‐linked Immunosorbent Assay: A Novel Platform for 8‐Hydroxy‐2'‐deoxyguanosine Biomarker Detection

Among the various oxidative DNA lesions, 8‐hydroxy‐2'‐deoxyguanosine (8‐OHdG) is widely recognized as a critical biomarker for assessing DNA damage and disease risk. Traditional detection methods, such as high‐performance liquid chromatography (HPLC), LC‐tandem mass spectrometry, and immunohistochemical techniques, provide high accuracy but suffer from limitations like time‐consuming procedures, high costs, and extensive sample preparation requirements. Enzyme‐linked immunosorbent assay (ELISA), a widely used and cost‐effective alternative, has been increasingly explored for 8‐OHdG detection. In this study, biotinylated antibodies (Abs), 8‐OHdG polyclonal Abs (pAbs), and horseradish peroxidase (HRP)‐containing nanoflowers (NFs) were synthesized and characterized using scanning electron microscopy, energy dispersive X‐ray spectroscopy, Fourier‐transform infrared spectroscopy, X‐ray diffraction, and elemental mapping analyses. The encapsulation efficiencies of Biotin Ab‐NFs and 8‐OHdG pAbs/HRP‐NFs were found to be 95% and 97%, respectively. The synthesized nanoflowers were integrated into direct and sandwich ELISA systems, demonstrating a detection limit comparable to commercial ELISA kits. To simplify the test procedure, an alternative system was developed by incorporating 8‐OHdG pAbs and anti‐rabbit immunoglobulin G‐HRP within nanoflowers, eliminating the need for a secondary Ab. The results showed successful antigen‐Ab interaction, high binding efficiency, and improved detection performance. Overall, this study highlights the potential of NF‐based ELISA for rapid, cost‐effective, and sensitive detection of 8‐OHdG, making it a promising approach for biomarker‐based disease diagnostics.