Magnetic nanoparticles (MNPs) are widely used in the development of biosensors for nucleic acid detection. Efficient immobilization of bioreceptors, such as DNA enzymes (deoxyribozymes, Dz), on the surface of MNPs is critical for sensor sensitivity. The aim of this work is to compare the conjugation efficiency of 10-23 deoxyribozyme with two widely used types of MNPs: streptavidin-coated (Strep-MNPs) and carboxyl-functionalized (COOH-MNPs).

We used biotin-modified (biotin-Dz) and amino-modified (NH₂-Dz) 10-23 Dz. The linear fluorescence range (λ_ex/λ_em = 480/525 nm) versus Dz concentration (0–1000 pM) was established by cleavage of a fluorogenic substrate (200 nM) in Col buffer (55°C, 1 h and 3 h). Conjugation of 20 nM Dz with Strep-MNPs (1 mg/mL) was performed in Col buffer (60 min, RT), with unbound Dz removed by washing (3x, Col buffer, magnetic separation). Conjugation of NH₂-Dz with COOH-MNPs was performed after EDC/NHS activation in Col buffer (30 min, RT), followed by Dz addition (2 h) and washing. A TTT oligonucleotide served as a non-specific binding control. After conjugation and washing, 200 nM substrate was added to MNPs, incubated for 1 h and 3 h (55°C), and fluorescence was measured. Conjugation efficiency was calculated as the activity of immobilized Dz (via calibration curve) relative to the activity of the initial 20 nM Dz solution.

Linear fluorescence ranges were as follows: biotin-Dz (0–50 pM, 1 h; 0–100 pM, 3 h); NH₂-Dz (0–1000 pM, 1 h; 0–100 pM, 3 h). After 1 h substrate incubation, we obtained the following percentages: biotin-Dz/Strep-MNPs: 0.32%; NH₂-Dz/COOH-MNPs: 0.07%. After 3 h, activity decreased to 0.02% for biotin-Dz/Strep-MNPs. The TTT conjugation efficiency was negligible (<0.01%).

Conjugation of 10-23 Dz using specific biotin–streptavidin binding (Strep-MNPs) was ~4.6 times more efficient than covalent amide binding (COOH-MNPs) under the tested conditions. Activity reduction during prolonged incubation may result from Dz inactivation. Streptavidin-coated MNPs are a preferred carrier for immobilizing biotin-modified 10-23 deoxyribozyme compared to carboxyl-MNPs. These findings are essential for developing high-performance Dz-based biosensors using magnetic nanoparticles.

This study was supported by grant FSER-2025-0019.