Various methods and carriers are routinely used for the separation of biopolymer molecules, such as mono- and multi-phosphorylated peptides or recombinant peptides/proteins with a polyhistidine tag (His-tag). However, these methods and carriers have limiting characteristics, for example, insufficient purity of separated proteins, limited surface area of microspheres accessible for binding, gradual release of the carrier ions during separation, poor mechanical stability, and toxicity of the released metal ions.
To address the needs for purification and enrichment of phosphopeptides and other biomolecules with high selectivity and simplicity of operation, a high surface area interface, based on anodic one-dimensional (1D) TiO2 nanotubes homogeneously decorated by Fe3O4 nanoparticles (TiO2NTs@Fe3O4NPs), and a tailored isolation protocol have been developed.
The 1D TiO2 nanotubes, when decorated with magnetite nanoparticles, seem to be a great candidate for purification of biomolecules for use in life science and biomedicine, fulfilling all requirements for high specificity, high surface area, proper magnetic functionality, and regeneration by photocatalytic means.
The combination of TiO2NTs@Fe3O4NPs with tailored isolation protocol offers an enhanced separation specificity, enrichment ability and possibility to widen the range of identified peptides as compared to the established TiO2 microspheres and Immobilized Metal Affinity Chromatography (IMAC)-based protocols.
The presented material (TiO2NTs@Fe3O4NPs) possesses several unique advantages: it is nontoxic, robust, it can be easily separated from any solution due to its intrinsic magnetism, and being based on TiO2, it can be simply decontaminated by UV-light-induced photocatalytic treatment at low costs and reused again at the same quality.
Our results have proved that the material retains almost exclusively phosphopeptides and shows 50% better preferential affinity for double or triple phosphorylated peptides. Moreover, it retains high number of unique phosphopeptides impossible to be isolated by other techniques. This attribute is fully exploitable mainly for analysis of hyperphosphorylated proteins associated with serious degenerative diseases.
Characteristics of TiO2NTs@Fe3O4NPs:
- TiO2 nanotubes with adjustable dimensions (length, diameter, wall thickness): length ≈ 5µm, inner diameter ≈ 230nm
- Surface modification using magnetic oxides (Fe3O4, Fe2O3, NiO, etc.) = easy handling; Fe3O4nanoparticles size - Ø 8 nm
- Adjustable structure (amorphous, anatase, rutile)
- Defined diffusion path for the reaction species accessibility of interiors
- pH stability: pH 1-12, stable in 5% trifluoroacetic acid and/or in organic solvent, e.g. acetonitrile
- Recyclability: minimum 4 times
- Chemical stability: all commonly used buffers and solvents
- Storage: dry or in water/organic solvent
- Production in lab from 100 mg up to 1g per day, batch to batch high reproducibility
The production of highly pure biomolecules, for example, proteins, polypeptides, oligosaccharides, or nucleic acids, is a key requirement for their use in medicine and life sciences. In particular, they are widely used for in vivo applications, such as for the production of efficient and selective biopharmaceuticals, including targeted bioactive therapeutics, recombinant proteins, or vaccines. The development of suitable materials and purification methods for these biologically active compounds is an important topic in current biomedical research.
The specificity of TiO2NTs@ Fe3O4NPs, surmounted by their unique properties, may open new pathways for the isolation and identification of clinically important biomolecules and for a whole range of in vitro life science applications.