Abstract
Despite the potential of antibody-coated nanoparticles (Ab-NPs) in many biological applications, there are very few successful, commercially available examples in which the carefully engineered nanomaterial has made it beyond the laboratory bench. Herein we explore the robustness and cost of protein-nanoparticle conjugation. Using multivalent polyamidoamine (PAMAM) dendrimers and dextran as crosslinkers, it was possible to retain colloidal stability during (i) NP-linker binding and (ii) the subsequent conjugation reaction between linker-coated NPs and proteins to generate monodisperse Ab-NPs. This was attributed to the physicochemical properties of the linkers, which were inherited by the NPs and thus benefited colloidal stability. Attaching negatively charged, EDC/sulfo-NHS-activated PAMAM to the NPs contributed to overall negative charge of particles, and in turn led to high electrostatic attraction between the protein and PAMAM-coated NPs during the reaction conditions. In contrast, using an uncharged, EDC/NHS-activated PAMAM dendrimer led to NP aggregation and lower protein binding efficiency. Dextran as a cost-effective, uncharged macromolecule allowed for steric repulsions between neighbouring particles during protein binding, thus inducing NP stability in solution, and also produced monodisperse Ab-NPs. By freeze-drying Ab-NPs from a 1% BSA solution it is possible to reconstitute the solid-form colloid back to a stable state by adding solvent and simply shaking the sample vial by hand. The consequences of the different surface chemistries and freeze-drying stabilizers on the colloidal stability of the NPs were probed by dynamic light scattering. The performance of Ab-NPs was compared in a simple fluorescence linked immunoassay in whole serum. Interestingly, the signal-to-noise ratios were similar for Ab-NPs using PAMAM and dextran, despite dextran binding fewer Abs per NP. We believe this work provides researchers with the tools and strategies for reliably generating Ab-NPs that can be used for a variety of biological applications.
Highlights
IntroductionPaper generating colloidally stable antibody-coated uorescent silica nanoparticles (Ab-NP) through the use of multivalent linker molecules
The rapid growth in nanotechnology research over the past two decades has led to the development of a ‘nano-Particle Zoo’, Journal of Materials Chemistry BPaper generating colloidally stable antibody-coated uorescent silica nanoparticles (Ab-NP) through the use of multivalent linker molecules.The most common method of antibody-nanoparticle conjugation is through covalent bond formation via a linker molecule
We hypothesized that the activation of PAMAM dendrimers with EDC/NHS renders it neutral, and upon attachment to the plain NP surface its neutrality is inherited by the negatively charged NPs, lowering the stability of the resultant NP-linker colloidal system
Summary
Paper generating colloidally stable antibody-coated uorescent silica nanoparticles (Ab-NP) through the use of multivalent linker molecules. The most common method of antibody-nanoparticle conjugation is through covalent bond formation via a linker molecule. The use of a linker creates a space between the protein and the nanoparticle surface, avoiding biomolecule denaturation or undesirable biomolecule–nanoparticle interactions. One of the major problems with the published protocols describing the bioconjugation reactions with nanoparticles is that they ignore the effect of the crosslinker introduction, which alters the surface chemistry of a given colloid and o en leads to particle aggregation. Bagwe et al.[10] for example, preserved the colloidal stability of silica nanoparticles by coating their surface with ‘chemically inert’, negatively charged phosphonates alongside ‘reactive’ –NH2 groups. There has, been little focus on how the characteristics of the crosslinker in uence nanoparticle stability, and whether this, in turn, can play a role in antibody binding efficiency and Ab-NP polydispersity
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