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Biotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) or biotin carboxyl carrier protein (BCCP) on the surface of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. BAP-displaying BacMPs (BAP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. Then the in vitro biotinylation of BAP-BacMPs was confirmed using alkaline phosphatase-labeled antibiotin antibody. In contrast, BacMPs displaying the intact 149 residues of AMB-1 BCCP (BCCP-BacMPs) and displaying the COOH-terminal 78 residues of BCCP (BCCP78-BacMPs) were biotinylated in AMB-1 cells. The in vivo biotinylation of BCCP-BacMPs and BCCP78-BacMPs was thought to be performed by endogenous AMB-1 biotin ligase. Streptavidin was introduced onto biotinylated BacMPs by simple mixing. In an analysis using tetramethyl rhodamine isocyanate-labeled streptavidin, approximately 15 streptavidin molecules were shown to be immobilized on a single BCCP-BacMP. Furthermore, gold nanoparticle-BacMP composites were constructed via the biotin-streptavidin interaction. The conjugation system developed in this work provides a simple, low-cost method for producing biotin- or streptavidin-labeled magnetic nanoparticles. Various functional materials can be site selectively immobilized on these specially designed BacMPs. By combining the site-selective biotinylation technology and the protein display technology, more innovative and attractive magnetic nanomaterials can be constructed.

Magnetic beads have served as a conventional bioassay platform in biotechnology. In this study, a fully automated immunoassay was performed using novel nano- and microbead-composites constructed by assembling nano-magnetic beads onto polystyrene microbeads, designated 'Beads on Beads'. Nano-sized bacterial magnetic particles (BacMPs) displaying the immunoglobulin G (IgG)-binding domain of protein A (ZZ domain) were used for the construction of 'Beads on Beads' via the interaction of biotin-streptavidin. The efficient assembly of 'Beads on Beads' was performed by gradual addition of biotin-labeled BacMPs onto streptavidin-coated polystyrene microbeads. Approximately 2000 BacMPs were uniformly assembled on a single microbead without aggregation. The constructed 'Beads on Beads' were magnetized and separated from the suspension by using an automated magnetic separation system with a higher efficiency than BacMPs alone. Furthermore, fully automated detection of prostate-specific antigens was performed with the detection limit of 1.48 ng mL(-1). From this preliminary assay, it can be seen that 'Beads on Beads' could be a powerful tool in the development of high-throughput, fully automated multiplexed bioassays.