RESUMO
Many biomedical and biosensing applications require functionalization of surfaces with proteins. To this end, the E/K coiled-coil peptide heterodimeric system has been shown to be advantageous. First, Kcoil peptides are covalently grafted onto a given surface. Ecoil-tagged proteins can then be non-covalently captured via a specific interaction with their Kcoil partners. Previously, oriented Kcoil grafting was achieved via thiol coupling, using a unique Kcoil with a terminal cysteine residue. However, cysteine-terminated Kcoil peptides are hard to produce, purify, and oxidize during storage. Indeed, they tend to homodimerize and form disulfide bonds via oxidation of their terminal thiol group, making it impossible to later graft them on thiol-reactive surfaces. Kcoil peptides also contain multiple free amine groups, available for covalent coupling through carbodiimide chemistry. Grafting Kcoil peptides on surfaces via amine coupling would thus guarantee their immobilization regardless of their terminal cysteine's oxidation state, at the expense of the control over their orientation. In this work, we compare Kcoil grafting strategies for the subsequent capture of Ecoil-tagged proteins, for applications such as surface plasmon resonance (SPR) biosensing and cell culture onto protein-decorated substrates. We compare the "classic" thiol coupling of cysteine-terminated Kcoil peptides to the amine coupling of (i) monomeric Kcoil and (ii) dimeric Kcoil-Kcoil linked by a disulfide bond. We have observed that SPR biosensing performances relying on captured Ecoil-tagged proteins were similar for amine-coupled dimeric Kcoil-Kcoil and thiol-coupled Kcoil peptides, at the expense of higher Ecoil-tagged protein consumption. For cell culture applications, Ecoil-tagged growth factors captured on amine-coupled monomeric Kcoil signaled through cell receptors similarly to those captured on thiol-coupled Kcoil peptides. Altogether, while oriented thiol coupling of cysteine-terminated Kcoil peptides remains the most reliable and versatile platform for Ecoil-tagged protein capture, amine coupling of Kcoil peptides, either monomeric or dimerized through a cysteine bond, can offer a good alternative when the challenges and costs associated with the production of monomeric cysteine-tagged Kcoil are too dissuasive for the application.
RESUMO
We compared different biofunctionalization strategies for immobilizing trastuzumab, an IgG targeting the HER2 biomarker, onto 100 nm spherical gold nanoparticles because of the E/K coiled-coil peptide heterodimer. First, Kcoil peptides were grafted onto the gold surface while their Ecoil partners were genetically encoded at the C-terminus of trastuzumab's Fc region, allowing for a strong and specific interaction between the antibodies and the nanoparticles. Gold nanoparticles with no Kcoil peptides on their surface were also produced to immobilize Ecoil-tagged trastuzumab antibodies via the specific adsorption of their negatively charged Ecoil tags on the positively charged gold surface. Finally, the nonspecific adsorption of wild-type trastuzumab on the gold surface was also assessed, with and without Kcoil peptides grafted on it beforehand. We developed a thorough workflow to systematically compare the immobilization strategies regarding the stability of nanoparticles, antibody coverage, and ability to specifically bind to HER2-positive breast cancer cells. All nanoparticles were highly monodisperse and retained their localized surface plasmon resonance properties after biofunctionalization. A significant increase in the amount of immobilized antibodies was observed with the two oriented coil-based strategies compared to nonspecific adsorption. Finally, all biofunctionalization strategies allowed for the detection of HER2-positive breast cancer cells, but among the investigated approaches, we recommend using the E/K coiled-coil-based strategy for gold nanoparticle biofunctionalization because it allows for the qualitative and quantitative detection of HER2-positive cells with a higher contrast compared to HER2-negative cells.