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

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

Development of a magnetic immunosorbent for on-chip preconcentration of amyloid β isoforms: Representatives of Alzheimer’s disease biomarkers

Determination of amyloid β (Aβ) isoforms and in particular the proportion of the Aβ 1-42 isoform in cerebrospinal fluid (CSF) of patients suspected of Alzheimer’s disease might help in early diagnosis and treatment of that illness. Due to the low concentration of Aβ peptides in biological fluids, a preconcentration step prior to the detection step is often necessary. This study utilized on-chip immunoprecipitation, known as micro-immunoprecipitation (μIP). The technique uses an immunosorbent (IS) consisting of magnetic beads coated with specific anti-Aβ antibodies organized into an affinity microcolumn by a magnetic field. Our goal was to thoroughly describe the critical steps in developing the IS, such as selecting the proper beads and anti-Aβ antibodies, as well as optimizing the immobilization technique and μIP protocol. The latter includes selecting optimal elution conditions. Furthermore, we demonstrate the efficiency of anti-Aβ IS for μIP and specific capture of 5 Aβ peptides under optimized conditions using various subsequent analytical methods, including matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), capillary electrophoresis, microchip electrophoresis, and immunoblotting. Synthetic Aβ peptides samples prepared in buffer and spiked in human CSF were analyzed. Finally, on-chip immunoprecipitation of Aβ peptides in human CSF sample was performed