The success of microfluidic immunocapture based on magnetic beads depends primarily on a sophisticated microscale separation system and on the quality of the magnetic immunosorbent. A microfluidic chip containing a magnetically stabilized fluidized bed (μMSFB), developed for the capture and on‐chip amplification of bacteria, was recently described by Pereiro et al.. The present work shows the thorough development of anti‐Salmonella magnetic immunosorbents with the optimal capture efficiency and selectivity. Based on the corresponding ISO standards, these parameters have to be high enough to capture even a few cells of bacteria in a proper aliquot of sample, e.g. milk. The selection of specific anti‐Salmonella IgG molecules and the conditions for covalent bonding were the key steps in preparing an immunosorbent of the desired quality. The protocol for immunocapturing was first thoroughly optimized and studied in a batchwise arrangement, and then the carrier was integrated into the μMSFB chip. The combination of the unique design of the chip (guaranteeing the collision of cells with magnetic beads) with the advanced immunosorbent led to a Salmonella cell capture efficiency of up to 99%. These high values were achieved repeatedly even in samples of milk differing in fat content. The rate of nonspecific capture of Escherichia coli (i.e. the negative control) was only 2%.
Iron oxide based particles functionalized by bioactive molecules have been utilized extensively in biotechnology and biomedicine. Despite their already proven advantages, instability under changing reaction conditions, non-specific sorption of biomolecules on the particles' surfaces, and iron oxide leakage from the naked particles can greatly limit their application. As confirmed many times, surface treatment with an appropriate stabilizer helps to minimize these disadvantages. In this work, we describe enhanced post-synthetic surface modification of superparamagnetic microparticles varying in materials and size using hyaluronic acid (HA) in various chain lengths. Scanning electron microscopy, atomic force microscopy, phase analysis light scattering and laser diffraction are the methods used for characterization of HA-coated particles. The zeta potential and thickness of HA-layer of HA-coated Dynabeads M270 Amine were -50 mV and 85 nm, respectively, and of HA-coated p(GMA-MOEAA)-NH2 were -38 mV and 140 nm, respectively. The electrochemical analysis confirmed the zero leakage of magnetic material and no reactivity of particles with hydrogen peroxide. The rate of non-specific sorption of bovine serum albumin was reduced up to 50% of naked ones. The coating efficiency and suitability of biopolymer-based microparticles for magnetically active microfluidic devices was confirmed. Highlights: Post-synthetic surface modification of magnetic microparticles by hyaluronic acid Hyaluronic acid -polymer of unique physicochemical and biological characteristics ABSTRACTIron oxide based particles functionalized by bioactive molecules have been utilized extensively in biotechnology and biomedicine. Despite their already proven advantages, instability under changing reaction conditions, non-specific sorption of biomolecules on the particles' surfaces, and iron oxide leakage from the naked particles can greatly limit their application. As confirmed many times, surface treatment with an appropriate stabilizer helps to minimize these disadvantages.In this work, we describe enhanced post-synthetic surface modification of superparamagnetic microparticles varying in materials and size using hyaluronic acid (HA) in various chain lengths. Scanning electron microscopy, atomic force microscopy, phase analysis light scattering and laser diffraction are the methods used for characterization of HA-coated particles. The zeta potential and thickness of HA-layer of HA-coated Dynabeads M270Amine were -50 mV and 85 nm, respectively, and of HA-coated p(GMA-MOEAA)-NH 2 were -38 mV and 140 nm, respectively. The electrochemical analysis confirmed the zero leakage of magnetic material and no reactivity of particles with hydrogen peroxide. The rate of nonspecific sorption of bovine serum albumin was reduced up to 50% of naked ones. The coating efficiency and suitability of biopolymer-based microparticles for magnetically active microfluidic devices was confirmed.
Carbonyl-reducing enzymes are important in both metabolism of endogenous substances and biotransformation of xenobiotics. Because sufficient amounts of native enzymes must be obtained to study their roles in metabolism, an efficient purification strategy is very important. Oracin (6-[2-(2-hydroxyethyl)aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c] isoquinoline) is a prospective anticancer drug and one of the xenobiotic substrates for carbonyl-reducing enzymes. A new purification strategy based on molecular recognition of carbonyl-reducing enzymes with oracin as a ligand is reported here. The type of covalent bond, ligand molecules orientation, and their distance from the backbone of the solid matrix for good stearic accessibility were taken into account during the designing of the carrier. The carriers based on magnetically active microparticles were tested by recombinant enzymes AKR1C3 and CBR1. The SiMAG-COOH magnetic microparticles with N-alkylated oracin and BAPA as spacer arm provide required parameters: proper selectivity and specificity enabling to isolate the target enzyme in sufficient quantity, purity, and activity.
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