A MOF-embedded/G4-packaged electrochemical labeling strategy-based biosensor for the simultaneous detection of β-thalassemia mutations

Abstract

Non-invasive prenatal testing (NIPT) offers a promising avenue for early diagnosis of β-thalassemia through the analysis of cell-free fetal DNA (cffDNA). In this work, a multi-gene electrochemical biosensor was proposed for the simultaneous ultra-sensitive detection of β-thalassemia mutations CD_41/42 and CD₁₇, based on the combination of a novel MOF-embedded/G4-packaging electrochemical labeling strategy and catalytic hairpin assembly (CHA). Firstly, methylene blue (MB) and ferrocene (Fc) were intricately embedded within metal-organic frameworks (MOFs) and then integrated with G-quadruplexes (G4) to create an all-encompassing package that acts as electrochemical signal labels (MB-MOFs-G4 and Fc-MOFs-G4). This design not only enhances the immobilization amount of the electrochemical substances but also employs its folded structure to effectively prevent the leakage of MB (or Fc) within the MOF. By integrating CHA and specific recognition probes, the biosensor allows for the simultaneous ultra-sensitive electrochemical detection of multiple β-thalassemia mutation genes. The structure and material properties of MB/Fc-MOFs-G4 have been experimental conformed. Differential pulse voltammetry (DPV) experiments have also shown its superior electrochemical stability and enhanced signals. When targeting CD_41/42 and CD₁₇, the detection linear range was tested as 1.0–500.0 fM with detection limits of 0.025 fM (CD_41/42) and 0.097 fM (CD₁₇). This strategy allows for the early detection of multiple gene mutations associated with β-thalassemia and can be adapted to detect mutations related to other diseases by altering the templates.