Abstract
BackgroundMagnetosomes (also called bacterial magnetic nanoparticles; BMPs) are biomembrane-coated nanoparticles synthesized by magnetotactic bacteria (MTB). Engineered BMPs fused to protein A (termed ∆F-BMP-FA) bind antibodies (Abs) automatically, and thus provide a series of potential advantages. However, no report so far has systematically evaluated functional applicability of genetically engineered BMPs.ResultsWe evaluated properties of ∆F-BMP-FA, and developed/optimized culture methods for host strain Magnetospirillum gryphiswaldense ΔF-FA, ∆F-BMP-FA extraction conditions, conditions for Ab conjugation to ∆F-BMP-FA surface, and procedures for antigen detection using ∆F-BMP-FA/Ab complexes (termed BMP-A-Ab). Fed-batch culture for 36 h in a 42-L fermentor resulted in yields (dry weight) of 2.26 g/L for strain ΔF-FA and 62 mg/L for ∆F-BMP-FA. Optimal wash cycle number for ∆F-BMP-FA purification was seven, with magnetic separation following each ultrasonication step. Fusion of protein A to BMPs resulted in ordered arrangement of Abs on BMP surface. Linkage rate 962 μg Ab per mg ∆F-BMP-FA was achieved. BMP-A-Ab were tested for detection of pathogen (Vibrio parahaemolyticus; Vp) surface antigen and hapten (gentamicin sulfate). Maximal Vp capture rate for BMP-A-Ab was 90% (higher than rate for commercial immunomagnetic beads), and detection sensitivity was 5 CFU/mL. ∆F-BMP-FA also bound Abs from crude mouse ascites to form complex. Lowest gentamicin sulfate detection line for BMP-A-Ab was 0.01 ng/mL, 400-fold lower than that for double Ab sandwich ELISA, and gentamicin sulfate recovery rate for BMP-A-Ab was 93.2%.ConclusionOur findings indicate that engineered BMPs such as ∆F-BMP-FA are inexpensive, eco-friendly alternatives to commercial immunomagnetic beads for detection or diagnostic immunoassays, and have high Ab-conjugation and antigen-adsorption capacity.
Highlights
Research on nanomaterials and nanotechnology has expanded rapidly during the past two decades, because of the diverse applications of these materials in biomedical, agricultural, environmental, and physiochemical areas [1]
Among the BMP-associated proteins in MSR-1, MamF is the most stable, and its gene and related mutant strains are commonly used in BMP surface display systems [25]
In view of the ultrasonication steps involved in isolation and purification of ∆F-BMP-FA, we examined the possibility that the fusion protein or some part of it is dropped from BMPs during these steps, and affects the purification process
Summary
Research on nanomaterials and nanotechnology has expanded rapidly during the past two decades, because of the diverse applications of these materials in biomedical, agricultural, environmental, and physiochemical areas [1]. A variety of organisms and materials, including bacteria [4, 5], fungi [6, 7], yeast [8], plant extracts [9], and waste materials [10], have been utilized as eco-friendly precursors or processing tools for synthesis of nanoparticles with potential clinical or industrial applications. Most of these biological methods involve precipitation in specific matrices, some are based on simple chemical reactions [11]. No report so far has systematically evaluated functional applicability of genetically engineered BMPs
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