Detection of tetanus toxoid with iron magnetic nanobioprobe

Subject Areas : biophysics

farzaneh karkhaneh yousefi ¹ , ahmad molaeirad ² , Ziba Karimi sadr ³ , Adele Divsalar ⁴ , Parvaneh Maghami ⁵

1 - Institute for Convergence Science & Technology, Islamic Azad University Science and Research Branch, Tehran, Iran
2 - Institute for Convergence Science & Technology, Islamic Azad University Science and Research Branch, Tehran, Iran
3 - Institute for Convergence Science & Technology, Islamic Azad University Science and Research Branch, Tehran, Iran
4 - Faculty of Biological Science, Kharazmi University, Tehran, Iran
5 - Institute for Convergence Science & Technology, Islamic Azad University Science and Research Branch, Tehran, Iran

Received: 2024-05-04 Accepted : 2025-03-08 Published : 2025-09-09

Keywords: Iron Oxide Nanoparticles, Tetra Ethoxysilane, 3- Aminopropyl Tetra Ethoxysilane, Silanization, Sol-gel, Tetanus toxoid monoclonal antibody,

Abstract :

Introduction

Iron oxide nanoparticle-based bioprobes are highly suitable platforms for the early diagnosis of fatal infectious diseases like tetanus, due to their exceptional reactivity, biocompatibility, cost-effective production, and straightforward isolation under a magnetic field. This research focused on employing magnetic iron nanobiosensors to detect tetanus toxoid.

Materials and Methods: The initial step involved coating iron oxide nanoparticles with silane groups through the hydrolysis of tetraethoxysilane (TEOS). This was followed by the introduction of amine (NH₂) groups onto the silanized surface using (3-aminopropyl)triethoxysilane (APTES). Antibodies were subsequently immobilized onto the modified nanoparticles. The success of the silanization and antibody conjugation procedures was analyzed using Fourier-transform infrared (FT-IR) spectroscopy, Energy-dispersive X-ray spectroscopy (EDX), Vibrating-sample magnetometry (VSM), and the Sulforhodamine B (SRB) test. The biosensor's performance and specificity were ultimately assessed through UV/Vis spectroscopy, fluorescence spectroscopy, an agglutination assay, and an enzyme-linked immunosorbent assay (ELISA).

Results: FT-IR spectroscopy verified the successful formation of Si-O-Si and Si-O-Fe bonds, alongside the presence of TCT chloride and amine groups originating from the anti-tetanus toxin antibodies on the nanoparticle surface. EDX analysis corroborated the silanization process by identifying the existence of silicon (Si), nitrogen (N), and carbon (C) elements. VSM results confirmed that the magnetic saturation value of the nanoparticles remained adequately high for biological uses post-conjugation. Antibody immobilization resulted in an increased absorption intensity observed via UV/Vis spectroscopy. Fluorescence data indicated a rise in the signal intensity of the nanobioprobe when exposed to an antigen concentration of 10 ng/mL. Furthermore, the nanobioprobes exhibited agglomeration in the presence of the tetanus toxoid antigen. The specific formation of an antigen-antibody-nanoprobe complex was confirmed by detection with an HRP-conjugated antibody, validating the high specificity of the nanobioprobe.

Conclusion: The developed magnetic nanobioprobe for tetanus detection, which has a detection limit of 10 ng/mL for the target antigen, presents an efficient solution for integration into Lab-on-a-Chip (LOC) devices and microfluidic chips, enabling rapid diagnostics.

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