Chemical–Chemical Redox Cycle Signal Amplification Strategy Combined with Dual Ratiometric Immunoassay for Surface-Enhanced Raman Spectroscopic Detection of Cardiac Troponin I

Improving the sensitivity and reproducibility of surface-enhanced Raman spectroscopy (SERS) methods for the detection of bioactive molecules is crucial in biological process research and clinical diagnosis. Herein, we designed a novel SERS platform for cardiac troponin I (cTnI) detection by a chemical–chemical redox cycle signal amplification strategy combined with a dual ratiometric immunoassay. First, ascorbic acid (AA) was generated by enzyme-assisted immunoreaction with a cTnI-anchored sandwich structure. Then, oxidized 4-mercaptophenol (ox4-MP) was reacted with AA to produce 4-mercaptophenol (4-MP). Quantitative analysis of cTnI was realized by a Raman signal switch between ox4-MP and 4-MP. Specifically, AA could be regenerated by reductant (tris(2-carboxyethyl) phosphine, TCEP), which in turn produced more signal indicator 4-MP, causing significant signal amplification for cTnI analysis by SERS immunosensing. Moreover, a dual ratiometric-type SERS method was established with the intensity ratio I₁₀₇₇/I₈₂₂ and I₆₃₃/I₈₂₂, which improved the reproducibility of the cTnI assay. The excellent performance of the chemical–chemical redox cycle strategy and ratio-type SERS assay endows the method with high sensitivity and reproducibility. The linear ranges of cTnI were 0.001 to 50.0 ng mL^–1 with detection limits of 0.33 pg mL^–1 (upon I₁₀₇₇/I₈₂₂) and 0.31 pg mL^–1 (upon I₆₃₅/I₈₂₂), respectively. The amount of cTnI in human serum samples yielded recoveries from 89.0 to 114%. This SERS method has remarkable analytical performance, providing an effective approach for the early diagnosis of cardiovascular diseases, and has great latent capacity in the sensitive detection of bioactive molecules.