Scope and Limitations of Surface Functional Group Quantification Methods: Exploratory Study with Poly(acrylic acid)-Grafted Micro- and Nanoparticles (original) (raw)
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Biofunctionalized nanomaterials have been extensively studied as a tool for a wide range of applications in biomedical fields. Despite many existing strategies to conjugate proteins to colloidal particles, determining the grafting efficiency that is, the amount of protein conjugated to the surface of a nanoparticle (NP)remains challenging. Formulations for biomedical applications are subjected to strict constraints, and a lack of precise characterization can prevent otherwise promising formulations to be explored further. Here, we propose a simple approach to precisely measure the grafting efficiency of biological molecules on the surface of three types of widely used NPs: polymeric NPs, inorganic NPs, and metallic NPs. This approach relies on the simultaneous hydrolysis of the grafted protein and the NP degradation in acidic conditions, followed by a spectrophotometric quantification of primary amines in solution. This strategy can be applied to any type of protein and does not require any labeling agent. It can be performed in a high-throughput manner as a routine experiment and only requires a conventional oven and a microplate reader.
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Analytical Chemistry, 2011
We present a novel, simple, and fast colorimetric method to quantify the total number of carboxy groups on polymer microparticle and nanoparticle surfaces. This method exploits that small divalent transition metal cations (M 2þ = Ni 2þ , Co 2þ , Cd 2þ ) are efficiently bound to these surface functional groups, which allows their extraction by a single centrifugation step. Remaining M 2þ in the supernatant is subsequently quantified spectrophotometrically after addition of the metal ion indicator pyrocatechol violet, for which Ni 2þ was identified to be the most suitable transition metal cation. We demonstrate that the difference between added and detected M 2þ is nicely correlated to the number of surface carboxy groups as determined by conductometry, thereby affording a validated measure for the trueness of this procedure. The variation coefficient of ∼5% found in reproducibility studies underlines the potential of this novel method that can find conceivable applications for the characterization of different types of poly(carboxylic acid)-functionalized materials, e.g., for quality control by manufacturers of such materials.
Journal of Immunological Methods, 2009
Microtiter wells are commonly used for bioassays. The sensitivity of such an assay depends on several instrumental and biochemical parameters such as the signal-to-background ratio and nonspecific binding of the label molecules. In this study, we have investigated the possible effects of well surface defects, well edges and denaturation of capture antibodies on the assay sensitivity. We used internally dyed Eu(III) chelate polystyrene nanoparticles as high specific activity labels in a thyroid stimulating hormone (TSH) sandwich-type model assay. The nanoparticle labels provide a high signal-to-background ratio in assays but the major limiting factor of the assay sensitivity is nonspecific binding of the labels. In our model assay the capture monoclonal antibodies were immobilized on microtiter wells passively or through streptavidin (SA)-biotin linkage. At first, commercially manufactured microtiter well surfaces were probed with an atomic force microscopy and significant structural inhomogeneities were found. The nonspecific binding of the nanoparticle conjugates did not appear to follow any of the microtiter well surface defect patterns in a number of experiments. In addition, the microtiter well edges did not increase the nonspecific binding. Denaturation of capture antibodies on solid surfaces has been proposed to expose amino acid sequences promoting nonspecific binding. This was studied by intentionally denaturing the surface capture antibodies by heat, detergent or acid treatment prior to the assay. Although specific signal was almost entirely lost no significant effect on nonspecific binding was observed. The passively adsorbed antibodies denatured at lower temperatures than those captured through streptavidin-biotin linkage. Evidently, the additional protein (SA) layer protected the capture antibody from denaturation whereas the solid surface appeared to act as a "catalyst" making the passively adsorbed antibody more susceptible to denaturation.
Covalent binding of proteins to grafted plastic surfaces suitable for immunoassays
Journal of Immunological Methods, 1987
A method for the introduction of chemically reactive groups onto polymeric surfaces, suitable for immunoassays, is described. The method, referred to as grafting, uses gamma irradiation from a 6°C0 source to initiate the free radical reaction. Polystyrene and polyvinyl chloride surfaces were grafted with crotonic acid and characterized with ESCA. 2 nmol/cm 2 of carboxylic groups were added during the method. Increased hydrophilic properties of the carboxylated surfaces were recorded by contact angle measurements. The grafting reaction did not impair the optical quality of the polymers studied. Various proteins were covalently linked to the modified surfaces of microtiter plates and tubes by means of a water-soluble carbodiimide. A significantly enhanced total capacity and strength of binding to grafted surfaces was demonstrated as compared to passive adsorption of the proteins to untreated surfaces.
Antibody-based surfaces: Rapid characterization using two complementary colorimetric assays
Analytica Chimica Acta, 2011
Finding a general solution for optimizing the grafting of antibody on solid surfaces is difficult due to the variety of material, grafting principles and chemistries or surface formats available (beads, microplates, fibers, etc.). Pre-screening methods able to assess grafting efficiency (GE) and specific activity (SA) are required. In this context, we present here two colorimetric assays that can be used on a wide variety of surface format, chemistry, etc. The first one, ADECA (Amino Density Estimation by Colorimetric Assay) allows a rapid estimation of grafted antibodies and allows calculating the GE. The second one, A 2 HRP (Antibody Anti-HorseRadish Peroxidase) provides a measure of the amount of active antibody, which, combined to ADECA, is used to determine the SA of grafted antibody. Analytical parameters (limit of detection, repeatability, linearity, etc.) of these two colorimetric assays are presented. Using two commercially available microplates, we demonstrated that, when used in parallel, these rapid and sensitive methods are well adapted to pre-screening of antibody grafting performances.