Methylene Blue-Loaded Nanocomposites Based SERS Immunoassay Sensor for Highly Sensitive NMP22 Detection

Nuclear matrix protein 22 (NMP22) is a crucial biomarker for bladder cancer (BC) diagnosis. However, its detection remains challenging due to the limitations of conventional diagnostic methods, including high costs, complex operations, insufficient sensitivity, and long analysis time. To address these issues, we developed an advanced SERS immunoassay sensor that combines magnetic Fe3O4nanocomposites (Fe3O4@Ab) and MB@Ab (methylene blue with antibody) nanocomposites for highly sensitive NMP22 detection. The Fe3O4magnetic nanocomposites enable rapid target separation through their superparamagnetic properties. The MB@Ab nanocomposites utilize an organic-inorganic hybrid structure to achieve high-density loading of Raman-active MB molecules and simultaneously synthesized silver colloid as SERS enhancement substrate, thereby significantly enhances the detected Raman signals to improve detection sensitivity of NMP22. The proposed SERS immunoassay sensor achieves high detection sensitivity with a detection limit of 0.34 pg/mL and excellent detection specificity for NMP22 among common tumor markers and interfering substances in urine. Experimental validation using human urine samples confirmed the stable performance in complex matrices with high reproducibility. The proposed SERS immunoassay sensor, with its exceptional sensitivity, ease of use, excellent repeatability, reliable clinical validation for NMP22 detection and potential adaptability to other tumor markers, demonstrates its significant advantages in early BC diagnosis and also holds great potential for widespread applications of diagnosing other tumors.