Protein self-assembly via Zr4+ ions on spore-based microspheres for immunoassays

- A biological method was presented to prepare monodisperse microspheres.
- Zr4+ ions were used as a universal linker to immobilize proteins onto the microspheres.
- This method is simple, low cost, high-performance, and eco-friendly.
- These assays were applied to the clinical diagnosis of M. bovis infection events.

Spore-based microspheres have ideal physical and chemical properties, which include various functionalities, excellent mechanical strength, uniform size distribution, high dispersity, a higher density than water, and a dimension of approximately 1 μm. The efficient surface immobilization of proteins is a key step in the development of microsphere-based immunosensors. In this study, we developed a novel method using Zr4+ ions as a high-performance and universal linker to immobilize proteins onto microsphere surfaces. Two different immunological test strategies were selected as models for the detection of Mycobacterium bovis infection events to illustrate the versatility of the proposed system: (1) Capture antigen proteins are linked to the surfaces of spore-based microspheres to detect M. bovis antibodies in bovine sera using flow cytometry; and (2) anti-bovine IFN-γ is immobilized on microsphere surfaces to measure the IFN-γ released by peripheral mononuclear blood cells from animals infected with M. bovis using the fluorescent horseradish peroxidase-H2O2-Amplex Rad reaction. These assays were successfully applied to the clinical diagnosis of M. bovis infection events. In the future, spore@Zr4+ particles can be used as a universal tool to detect a broad range of analytes.

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