Integrating Aptasensor With Mass-Tag Signal Amplification Strategy for the Detection of Three Steroid Hormones in Serum

Rationale Steroid hormone testing is critical for assessing endocrine function, diagnosing related disorders, and monitoring therapeutic efficacy. However, current mainstream detection methods have limitations. Although liquid chromatography–tandem mass spectrometry (LC–MS/MS), regarded as the gold standard, offers high sensitivity and specificity, it involves complex and time-consuming procedures. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are simple and fast but are limited by poor throughput for multitarget detection. Therefore, it is crucial to develop an analytical method that streamlines procedures and enables efficient parallel detection of multiple targets. Methods We developed an integrated signal-enhanced aptasensor platform for pooled MS detection of three steroid hormones in serum. It combines aptamer recognition with mass-tag amplification. This approach uses biorecognition instead of chromatography, while mass spectrometry enables simultaneous readout of multiple mass tags from combined samples. Results The method was evaluated by detecting three steroid hormones (vitamin D, cortisol, and testosterone) in simulated serum samples and human serum samples. The limits of detection (LODs) for these analytes ranged from 0.411 to 6.796 nM, which are below the established clinical cut-off values for each steroid, demonstrating the requisite sensitivity for detection. Conclusions This integrated signal-enhanced aptasensor outperforms conventional LC–MS/MS in efficiency and ELISA in throughput, enabling the quantification of multiple serum steroid hormones. Therefore, we believe that this method could be potentially useful in the clinical screening of hormone-related disorders and suitable for the analysis of serum.