Developing G value as an indicator for assessing the molecular status of immobilized antibody.

Antibody-functionalized nanoparticles (Ab-NPs) are widely used in bioassays due to their excellent affinity, specificity toward antigen, and ease of operation. However, the uncontrollable molecular status of antibodies on NPs severely limits their applications. This work aims at developing a simple method to evaluate the antigen-binding activity of Ab-NPs using two parameters, i.e., antibody adsorption amount and antigen-binding strength. Herein, we proposed a mathematical expression, G, to quantitively describe the amount and strength of Ab-NPs. G value could be used to assess the antigen-binding performance of NPs influenced by surface and solution factors. Seven types of polymers with different surface properties, including four positively and three negatively charged polymer brushes, were grown from silica NPs via surface-initiated atom transfer radical polymerization (SI-ATRP). A pair of antigen and antibody, human chorionic gonadotropin (hCG) and anti-hCG, were selected to screen the antibody immobilization property of polymer brushes. Among them, the G values of 2 polymer-NPs with opposite charges reached maximum, resulting in low detection limits for hCG, where pDMAEA-NP and pMMA-NP represent Poly[N,N-(dimethylamino)ethyl acrylate]-NP and poly(methyl methacrylate)-NP, respectively. The G value of Ab-NPs makes it feasible to estimate the molecular status of the adsorbed antibodies on surfaces, thus showing great potential for in vitro biosensing and bioseparation.