Hybrid Nanoflower-enhanced Enzyme-linked Immunosorbent Assay: A Novel Platform for 8-Hydroxy-2'-deoxyguanosine...

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.