New applications of Affimer® proteins in nanoparticle production

Magnetic nanoparticles have applications in fields as diverse as high density data storage, waste water treatment and cancer therapy, but to manufacture these particles with sufficient uniformity requires high temperatures and expensive reagents. A study (link is external) from the University of Sheffield published in Chemical Science, demonstrates how Affimer technology can be applied to develop a suite of magnetite-interacting proteins that can direct the morphology of nanoparticles without the need for adverse reaction conditions.

TEM analysis of magnetite nanoparticles shows that including Affimer proteins in the reaction produced particles with increased cubic and angular morphology (A) compared to controls (B).
TEM analysis of magnetite nanoparticles shows that including Affimer proteins in the reaction produced particles with increased cubic and angular morphology (A) compared to controls (B).

Magnetotactic bacteria use nucleating proteins to synthesise highly uniform magnetite crystals under ambient conditions, in shapes and sizes that are not possible in synthetic manufacturing. While using bacterially isolated proteins may offer the opportunity to achieve crystals with a uniform morphology and a narrow size distribution, these proteins are often difficult and expensive to produce and purify. As magnetic nanoparticles are important for many different purposes, easier and cheaper production methods are of benefit to a number of industries. 

To overcome these production issues a team of researchers, led by Dr Sarah Staniland from the University of Sheffield, used Affimer binders to fast-track the identification of proteins that can drive the process of uniform magnetite nanoparticle formation. Using cubic magnetite nanoparticles the researchers screened the Affimer library to identify Affimer proteins that could bind to specific planes of these nanoparticles.
ELISAs were used to assess the binding of 72 Affimer proteins to cubic magnetite nanoparticles and the 48 Affimer proteins with the highest affinity were selected for sequence analysis. The Affimer binders isolated showed a preference for basic residues within the first variable binding loop, with the predominant selection of histidine (9.3%) and lysine (26.4%) residues and almost total exclusion of acidic residues. The high presence of lysine residues within the Affimer protein binding site led to initial purifications of the Affimer binders being contaminated with nucleic acids. This was easily corrected using a heparin resin to remove the DNA and RNA, but suggests that it might be possible to isolate Affimer proteins targeted to DNA-binding applications.

The Affimer proteins selected from the screening study showed a high level of specificity to the magnetite target material, as they showed a high affinity for magnetite, but no interaction with the control zinc oxide by ELISA. Inclusion of the Affimer reagents in magnetite nanoparticle precipitation reactions gave no apparent increase in nanoparticle size. However, TEM analysis of the particles created in the presence of the Affimer proteins showed more regularity in their structure, being more cubic and angular than particles from control reactions. The addition of sufficient Affimer protein concentration to these nanoparticle production reactions also eliminated the presence of additional iron oxide materials often seen in these reactions. This demonstrates that the Affimers with affinity for the cubic magnetite nanoparticle structures appear to stabilise this structure during the precipitation reaction to promote the formation of cubic structures over other molecular assemblies.

Peptides of the Affimer protein’s variable loop sequence were added to the magnetite precipitation reactions to assess their ability to promote cubic crystal formation in the same way as the Affimer proteins. Variable crystal shapes resulted, indicating that the Affimer protein scaffold is essential to constrain the variable peptide to allow its function in cubic magnetite nanoparticle production.

This exciting study demonstrates the power of Affimer proteins being applied to material science, and shows their potential to power a process that typically requires extreme reaction conditions under standard ambient conditions. Affimer reagents offer a promising new approach to the precision manufacture of magnetite nanoparticles.