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

Spacer Control the Dynamic of Triplex Formation between Oligonucleotide-Modified Gold Nanoparticles

A novel method was developed to control the dynamic of triplex formation between oligonucleotide-modified gold nanoparticles in the presence of complementary strand. The solution containing the oligonucleotide 5′-SH-ACA CAC ACA CAC CTT TCT TTC CTT TCT TTC-3′(oligo-1)-modified gold nanoparticles was red in color. Due to triplex formation, there was a tiny change in color on addition of the complementary oligonucleotide 5′-GAA AGA AAG GAA AGA AAG-3′(oligo-3). The addition of oligonucleotide 5′-GTG TGT GTG TGT-3′(oligo-2) induced the spacer portion of oligo-1 to change from single strand to rigid duplex structure and protrude from the surface of the gold colloid, removing the physisorption between oligo-1 and the gold nanoparticles successfully. Therefore, when the oligo-2 was added accompanied by oligo-3 at pH 5.6 and 6.0 μM spermine, larger aggregates were formed and the color of the solution changed from red to blue within 20 min. The oligo-2 hybridized with the spacer portion of oligo-1 and had no effect on the stability of triplex DNA; thereby, the melting temperatures of the triplex DNA were 51 and 53 °C in the absence and presence of oligo-2, respectively. Oligo-3 played a crucial role in the triplex formation between nanoparticles. When oligo-3 was replaced with 5′-GAA AGA AAG TAA AGA AAG-3′ (oligo-4, single-base mismatched) and 5′-GAA AGT AAG GAA TGA AAG-3′ (oligo-5, double-base mismatched), respectively, the melting temperature decreased from 53 to 41 °C and eventually to 33 °C