Strategies for the development of an electrochemical bioassay for TNF-alpha detection by using a non-immunoglobulin bioreceptor

TNF-alpha is an inflammatory cytokine produced by the immune system. Serum TNF-alpha level is elevated in some pathological states such as septic shock, graft rejection, HIV infection, neurodegenerative diseases, rheumatoid arthritis and cancer. Detecting trace amount of TNF-alpha is, also, very important for the understanding of tumor biological processes. Detection of this key biomarker is commonly achieved by use of ELISA or cytofluorimetric based methods. In this study the traditional optical detection was replaced by differential pulse voltammetry (DPV) and an affinity molecule, produced by evolutionary approaches, has been tested as capture bioreceptor. This molecule, namely a combinatorial non-immunoglobulin protein (Affibody (R)) interacts with TNF-alpha selectively and was here tested in a sandwich assay format. Moreover magnetic beads were used as support for bioreceptor immobilization and screen printed carbon electrodes were used as transducers.TNF-alpha calibration curve was performed, obtaining the detection limit of 38 pg/mL, the quantification range of 76-5000 pg/mL and RSD%=7. Preliminary results of serum samples analysis were also reported. (C) 2016 Elsevier B.V. All rights reserved

Reversible immobilization of glycoamylase by a variety of Cu2+-chelated membranes

Glycoamylase (AMG) is an ?-amylase enzyme which catalyzes the breakdown of large a(1,4)-linked malto-oligosaccharides to glucose. It is an extracellular enzyme and is excreted to the culture medium. In this study, AMG was immobilized on a variety of metal affinity membranes, which were prepared by chelating Cu2+ ions onto poly(hydroxyethyl methacrylate) (PHEMA) using N-methacryloyl-(L)-histidine methyl ester (MAH), N-methacryloyl-(L)-cysteine methyl ester (MAC), and N-methacryloyl-(L)-phenylalanine methyl ester (MAPA) as metal-chelating comonomers for reversible immobilization of AMG. The PHEMAH, PHEMAC, PHEMAPA membranes were synthesized by UV-initiated photo-polymerization and Cu2+ ions were chelated on the membrane surfaces. Cu2+-chelated membranes were characterized by swelling tests, SEM, contact angle measurements, elemental analysis, and FTIR. AMG immobilization on the Cu2+-chelated membranes was performed by using aqueous solutions of different amounts of AMG at different pH values and Cu2+ loadings. Durability tests concerning desorption of AMG and reusability of the Cu2+-chelated membranes yielded acceptable results. It was computationally determined that AMG possesses four likely Cu2+/Zn2+ binding sites, away from the catalytic site, to which the metal-chelated membranes can be efficiently used. (C) 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 201

Molecularly imprinted composite cryogels for hemoglobin depletion from human blood

A molecularly imprinted composite cryogel (MICC) was prepared for depletion of hemoglobin from human blood prior to use in proteome applications. Poly(hydroxyethyl methacrylate) based MICC was prepared with high gel fraction yields up to 90%, and characterized by Fourier transform infrared spectrophotometer, scanning electron microscopy, swelling studies, flow dynamics and surface area measurements. MICC exhibited a high binding capacity and selectivity for hemoglobin in the presence of immunoglobulin G, albumin and myoglobin. MICC column was successfully applied in fast protein liquid chromatography system for selective depletion of hemoglobin for human blood. The depletion ratio was highly increased by embedding microspheres into the cryogel (93.2%). Finally, MICC can be reused many times with no apparent decrease in hemoglobin adsorption capacity. Copyright (C) 2014 John Wiley & Sons, Ltd

Synthesis and characterization of amino acid containing Cu(II) chelated nanoparticles for lysozyme adsorption

WOS: 000313155500073PubMed ID: 25428106Immobilized metal ion affinity chromatography (IMAC) is a useful method for adsorption of proteins that have an affinity for transition metal ions. In this study, poly( hydroxyethyl methacrylate-methacryloyl-L-tryptophan) (PHEMATrp) nanoparticles were prepared by surfactant free emulsion polymerization. Then, Cu(II) ions were chelated on the PHEMATrp nanoparticles to be used in lysozyme adsorption studies in batch system. The maximum lysozyme adsorption capacity of the PHEMATrp nanoparticles was found to be 326.9 mg/g polymer at pH 7.0. The nonspecific lysozyme adsorption onto the PHEMA nanoparticles was negligible. In terms of protein desorption, it was observed that adsorbed lysozyme was readily desorbed in medium containing 1.0 M NaCl. The results showed that the metal-chelated PHEMATrp nanoparticles can be considered as a good adsorbent for lysozyme purification. (C) 2012 Elsevier B.V. All rights reserved