A new homogeneous electrochemical immunoassay strategy was developed for ultrasensitive detection of carcinoembryonic antigen (CEA) based on target-induced proximity hybridization coupled with rolling circle amplification (RCA). The immobilization-free detection of CEA was realized by the use of an uncharged peptide nucleic acid (PNA) probe labeled with ferrocene (Fc) as the electroactive indicator on a negatively charged indium tin oxide (ITO) electrode. In the presence of a target protein and two DNA-labeled antibodies, the proximate complex formed in homogeneous solution could unfold the molecular beacon, and a part of the unfolded molecular beacon as a primer hybridized with the RCA template to initiate the RCA process. Subsequently, the detection probe modified Fc (Fc-PNAs) hybridized with the long amplified DNA products. The consumption of freely diffusible Fc-PNAs (neutrally charged) resulted in a significant reduction of the Fc signal due to the fact that long amplified DNA/Fc-PNA products were electrostatically repelled from the ITO electrode surface. The reduction of the electrochemical signal (signal-off) could indirectly provide the CEA concentration. Under the optimal conditions, CEA detection was implemented in a wide range from 1 pg mL to 10 ng mL, with a low detection limit of 0.49 pg mL. The proposed strategy exhibited advantages of good selectivity, high sensitivity, acceptable accuracy, and favorable versatility of analytes. Moreover, the practical application value of the system was confirmed by the assay of CEA in human serums with satisfactory results.
The DNA demethylation pathway has been discovered to play a significant role in DNA epigenetics. This pathway removes the methyl group from cytosine, which is involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) by ten-eleven translocation (TET) proteins. Then, 5-hmC can be iteratively oxidized to generate 5-formylcytosine (5-foC) and 5-carboxylcytosine (5-caC). However, 5-hmC, 5-foC, and 5-caC are hardly detected due to their low content. In this study, we have developed a LC-HRMS method coupled with derivatization to accurately and simultaneously quantify 5-mC levels, along with its oxidation products in genomic DNA. Derivatization was carried out using 4-dimethylamino benzoic anhydride, which has been shown to improve separation and enhance the detection sensitivity. Finally, we successfully applied this method towards the quantification of 5-mC, 5-hmC, 5-foC, and 5-caC in genomic DNA isolated from both human breast cancer tissue and tumoradjacent normal tissue. We show that 5-foC and 5-caC are increased in tumor tissue. In addition, the levels of 5-mC, 5-hmC, 5-foC, and 5-caC measured in tumor tissue versus tumor-adjacent tissue were found to be distinct among different classifications. This suggests that cytosine modifiers could be used as potential biomarkers for determining the stage of development of breast cancer, as well as prognosis.
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