Evaluation of Colorimetric BCA-Based Quantification of Hydrazide Groups on Magnetic Particles

Zuzana Svobodová ,Zdeněk Plichta ,Barbora Jankovičová ,Daniel Horák ,Zuzana Bı́lková

doi:10.1155/2018/5492893

Abstract

Magnetic micro- and nanoparticles (MPs) are considered to provide excellent solid support for many immunoanalytical and bioaffinity applications, particularly when they contain hydrazide groups available for site-specific immobilization of various glycoproteins, such as immunoglobulin G and enzymes. To prepare a highly active bioaffinity carrier with sufficient binding capacity, knowledge as to the type and concentration of functional groups used for ligand binding is crucial. Described here is a simple, nontoxic method for rapid estimation of hydrazide functional groups bound to MPs using bicinchoninic acid (BCA). BCA kits are routinely used for colorimetric detection and quantification of total protein in liquid samples. In this study, the BCA reagent was applied for quantification of hydrazide groups on MPs. The approach was carried out using an adipic acid dihydrazide (ADH) solution and subsequently using various hydrazide-containing magnetic and nonmagnetic carriers differing in the density of hydrazide groups. The BCA test’s results obtained on the MPs were compared with those from conventional amino and hydrazide group quantification by the 2,4,6-trinitrobenzenesulfonic acid (TNBS) test.

Highlights

Interest in covalent binding of proteins to planar or spherical solid supports is associated with the rapid development of new affinity carriers and enzyme catalysts [1]
The first experiments were designed to confirm the assumption that the bicinchoninic acid (BCA) reagent may be used to estimate the amount of hydrazide groups on micro- and nanoparticles (MPs)
A simple, one-step, rapid, low-toxic method for the quantitative determination of hydrazide groups on solid support using the BCA reagent was evaluated on various MPs, including macroporous bead cellulose, polymer MPs, and nonporous silica MPs

Summary

Introduction

Interest in covalent binding of proteins to planar or spherical solid supports is associated with the rapid development of new affinity carriers and enzyme catalysts [1]. One widely used coupling method involves site-specific immobilization of glycoproteins (enzymes, hormones, and/or antibodies) through their carbohydrate moieties on hydrazide solid supports [8, 9]. The fragment antigen-binding (so-called “Fab”) remain sterically available for specific reaction with the antigen as the target molecule [10]. The efficiency of such immunomagnetic carriers is considered to be nearly identical to that of biotinstreptavidin systems, yet they are simple and cost-effective. They are 2.5 times more efficient than is random coupling of IgG molecules via amino groups [11]

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Conclusion