This manuscript describes a novel method for the biofunctionalization of glass surfaces with polyhistidine-tagged proteins. The main innovation of this methodology consists of the covalent binding between the nitrilotriacetic acid (NTA) moiety and the proteins, ensuring not only orientation, but also stability of the recombinant proteins on NTA-covered surfaces. In this work, as C-terminal polyhistidine tagged cadherin extracellular fragments have been used, this methodology guarantees the proper orientation of these proteins, by mimicking their insertion into cell plasma membranes. These biofunctionalized surfaces have been characterized by confocal microscopy, X-ray photoelectron spectroscopy, contact angle, and atomic force microscopy, showing a high density of cadherins on the glass surfaces and the stability of the linkage. The prepared materials exhibited a high tendency to promote cell spreading, demonstrating the functionality of the protein and the high utility of these biomaterials to promote cell adhesion events. Interestingly, differences in the cytoskeleton organization have been observed in cells adhering to surfaces with no cadherins or with nonoriented cadherins, in comparison to surfaces functionalized with well-oriented cadherins. This method, which allows the robust immobilization of polyhistidine tagged proteins due to their covalent binding and with a defined orientation, may also find particular usefulness in the making of protein biochips, for analysis of protein-protein interactions, as well as structural and single-molecule studies.
Selecting the appropriate nanoparticle, functionalization chemistry and sensing methodology can speed up the translation of liquid biopsies into the clinic.
Biomimetic silica particles can be synthesized as a nanosized material within minutes in a process mimicked from living organisms such as diatoms and sponges. In this work, we have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our approach enables the formation of spheres with different physicochemical properties. Particles using BSA as a protein template were smaller (∼250–380 nm) and were more monodisperse than those lacking the proteic core (∼700–1000 nm) as seen by dynamic light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning electron microscopy (ESEM) analysis. The absence of BSA during synthesis produced silica nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas particles containing BSA developed porosity in the range of 4 to 5 nm which collapsed on the removal of BSA, thus producing smaller pores. These results were in accordance with the pore size calculated by high-resolution transmission electron microscopy (HTEM). The reproducibility of the BSA-templated nanoparticle properties was determined by analyzing four batches of independent synthesizing experiments that maintained their properties. The high positive superficial charge of the nanoparticles facilitated adsorption under mild conditions of a range of proteins from an E. coli extract and a commercial preparation of laccase from Trametes versicolor. All of the proteins were quantitatively desorbed. Experiments conducted showed the reusability of the particles as supports for the ionic adsorption of the biomolecules. The protein loading capacity of the BSA-based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg·g–1 of particles.
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