Development of Novel Magnetic Nanoparticles Modified with Carboxylic Groups for Nucleotide Sequence Immobilization and miRNA Detection

In this work, a simple and rapid strategy based on the use of an ultrasound probe for the uniform modification of magnetic nanoparticles (MNPs) with carboxylic groups is proposed for the covalent immobilization of nucleotide sequences and miRNA detection. The magnetic nanoparticles were successfully functionalized using citric acid (5 min), compared to other carboxylic compounds (maleic acid and EDTA). Additionally, they yielded higher immobilization rates compared to commercially available ones, which are often very expensive. The immobilization of the capture probe onto MNPs-COOH was carried out using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide as a carboxyl group activating agent. The hybridization of the target miRNA and recognition probe occurred simultaneously. A portion of the target miRNA hybridized with the recognition probe, while another portion of the target miRNA hybridized with the capture probe immobilized on MNPs-COOH. The recognized probe is detected by avidin-labeled alkaline phosphatase. The recognized probe was assessed based on the colorimetric para-nitrophenylphosphate response, generating a yellow color measurable at 405 nm. The colorimetric genosensor assay exhibited a linear range from 0.1 nM to 25 nM miRNA-222, with a detection limit of 0.08 nM of miRNA-222.