Mg²⁺-promoted high-efficiency DNA conjugation on polydopamine surfaces for aptamer-based ochratoxin A detection

Surface immobilization of DNA is the foundation of a broad range of applications in biosensing and specific DNA extraction. Polydopamine (PDA) coatings can serve as intermediate layers to immobilize amino- or thiol-labelled molecules, including DNA, onto various materials through Michael addition and/or Schiff base reactions. However, the conjugation efficiency is limited by electrostatic repulsion between negatively charged DNA and PDA. Recently, it has been reported that polyvalent metal ions (such as Mg²⁺ and Ca²⁺) can mediate the adsorption of DNA on PDA surfaces. Inspired by this, in this work we aimed to exploit polyvalent metal ions to facilitate the conjugation of DNA on PDA.

Mg²⁺ was used to promote the conjugation of amino-terminated DNA complementary to ochratoxin A (OTA) aptamer (cDNA-NH₂) on PDA-coated magnetic nanoparticles (Fe₃O₄@PDA). After the reaction, the unlinked cDNA-NH₂ adsorbed on Fe₃O₄@PDA mediated by Mg²⁺ was removed with EDTA. In the presence of 20 mM Mg²⁺, the amount of covalently linked cDNA-NH₂ increased approximately 11-fold compared to that in the absence of Mg²⁺. The resulting Fe₃O₄@PDA@cDNA conjugates exhibited superior hybridization capacity towards OTA aptamers, minimal nonspecific adsorption, and excellent chemical stability. The conjugates combined with fluorophore-labelled aptamers were employed for OTA detection, achieving a limit of detection (LOD) of 2.77 ng mL^-1. To demonstrate versatility, this conjugation method was extended to Ca²⁺-promoted conjugation of cDNA-NH₂ on Fe₃O₄@PDA nanoparticles and Mg²⁺-promoted conjugation of cDNA-NH₂ on PDA-coated 96-well plates.

The conjugation efficiency of DNA on PDA was significantly improved with the assistance of polyvalent metal ions (Mg²⁺ and Ca²⁺), providing a facile and efficient method for DNA immobilization. Due to the substrate-independent adhesion property of PDA, this method demonstrates versatility in DNA surface modification and holds great potential for applications in target extraction, biosensing, and other fields.