Bacteriophage Bundles with Pre-Aligned Ca Initiate the Oriented Nucleation and Growth of Hydroxylapatite

Abstract

Inorganic ions may direct the self-assembly of biomacromolecules into nanostructures which can further be used as a reactant and matrix for nanomaterials synthesis and assembly. Here we use bone mineral and filamentous bacteriophage as a model to demonstrate this concept. Divalent calcium ions are found to trigger the electrostatic self-assembly of anionic nanofiber-like bacteriophages into bundle structures where calcium ions are pre-organized between bacteriophage nanofibers. The resultant Ca(2+)-bacteriophage bundles can be separated and purified from the aqueous solution. The nanostructures of the bundles are verified by zeta potential analysis, small angle x-ray scattering and transmission electron microscopy. Because of the transcription of the bacteriophage chiral surface to the periodic alignment of pre-loaded Ca(2+), the Ca(2+)-bacteriphage bundles can serve as both Ca sources and biotemplates to initiate the oriented nucleation and growth of nanocrystalline hydroxyapatite in phosphate solution or in simulated body fluid. This work provides new insights into biomineralization and represents a new approach to the fabrication of biomolecular-inorganic hybrid layered nanostructures.

Figure 1

a) Low-magnification TEM images of fd-phage bundles (Ca²⁺-phage) induced by addition of CaCl₂ solution (0.2 M) to phage solution. b) High-magnification TEM images of the Ca²⁺-(fd)phage bundles. c) EDS analysis of the formed Ca²⁺-(fd)phage bundles, which shows the presence of Ca and P. d) ζ potential of fd phage in aqueous solution under different concentrations of Ca²⁺, which shows a rapid decrease of ζ potential with increasing concentration of Ca²⁺.

Figure 2

Left: Comparison of SAXS profiles of pure fd phage (bottom) and Ca²⁺-(fd)phage bundles (top) in water. The amplified version of the SAXS profile of pure fd phage (bottom) is shown in Figure S4. Right: diagram illustrating a Ca²⁺-(fd)phage bundle where phage and Ca²⁺ layer are organized into an ordered layer structure, which explains the new peak in the SAXS profile of the Ca²⁺-phage bundles (see text).

Figure 3

TEM images and corresponding SAED patterns of HAP-phage nanocomposites. (a) wider and (b) narrower HAP-fd phage bundles. Insets are the SAED patterns taken from the highlighted sections. (c) EDS analysis of the HAP-phage bundle in (a). It should be noted that phages in these images are not visible under TEM because they are not stained.

Scheme 1

Strategy for oriented nucleation and growth of HAP nanocrystals within aligned phage nanofibers. Ca²⁺ ions are first added to anionic phage solution to induce the formation of Ca²⁺-phage assembly (bundle). Then the Ca²⁺-phage bundles are purified by centrifugation and incubated in a phosphate solution to induce the oriented nucleation and growth of HAP within the channels of the bundles, resulting in the formation of HAP-phage assemblies.