Abstract
The utilization of immuno-magnetic nanoparticles (MNPs) for the selective capture, enrichment, and separation of specific glycoproteins from complicated biological samples is appealing for the discovery of disease biomarkers. Herein, MNPs were designed and anchored with abundant boronic acid (BA) and photoreactive alkyl diazirine (Diaz) functional groups to obtain permanently tethered Fc-fused Siglec-2 and antiserum amyloid A (SAA) mAb with the assistance of reversible boronate affinity and UV light activation in an orientation-controlled manner. The Siglec-2-Fc-functionalized MNPs showed excellent stability in fetal bovine serum (FBS) and excellent efficiency in the extraction of cell membrane glycoproteins. The anti-SAA mAb-functionalized MNPs maintained active Ab orientation and preserved antigen recognition capability in biological samples. Thus, the BA-Diaz-based strategy holds promise for the immobilization of glycoproteins, such as antibodies, with the original protein binding activity maintained, which can provide better enrichment for the sensitive detection of target proteins.
Highlights
Multifunctional metal nanoparticles (NPs), in particular superparamagnetic iron oxide NPs, have been used as biochemical sensors and biomolecular imaging contrast agents in extensive biomedical applications, including diagnostics, therapeutics, and theranostics.[1,2,3] A tunable core size, varying from a few up to tens of nanometers, and various shapes combined with the large surface area to volume ratio of magnetic nanoparticles (MNPs), make them suitable for high-capacity binding to proteins and cells.[4]
A recombinant fusion protein consisting of the human IgG1 Fc fragment fused with the extracellular domain of Siglec-2 (Siglec-2–Fc)[40] was prepared to facilitate carbohydrate–boronic acid (BA) interactions
In the presence of Siglec-2–Fc, the surface BAs on MNPs associate with proteins by targeting the N-glycan chain in the constant Fc region
Summary
Multifunctional metal nanoparticles (NPs), in particular superparamagnetic iron oxide NPs, have been used as biochemical sensors and biomolecular imaging contrast agents in extensive biomedical applications, including diagnostics, therapeutics, and theranostics.[1,2,3] A tunable core size, varying from a few up to tens of nanometers, and various shapes (spherical, hexagonal, and cubic) combined with the large surface area to volume ratio of magnetic nanoparticles (MNPs), make them suitable for high-capacity binding to proteins and cells.[4]. The nature of MNP surface chemistry promotes the modular and controlled conjugation of biomolecules, especially using amine and carboxyl functionalities.[15] Proteins adsorbed onto MNP surfaces through hydrophobic and polar interactions in random orientations are not sufficiently stable for many applications. One of the most popular methods for the covalent conjugation of MNPs with Abs involves carbodiimide-based coupling[6,9,10,11,12,13] between carboxyl groups on the MNP and the most reactive side chain 3-amines of lysine residues of the Ab, which are positioned at various locations on the surface of the Ab. Despite the lack of Ab modi cation, this strategy can 8600 | Chem.
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