Recovery of immunoglobulin G from rabbit serum using κ-carrageenan-modified hybrid magnetic nanoparticles.

Immunoglobulin G (IgG) has been used in the treatment of cancer, autoimmune diseases and neurological disorders, however, the current technologies to purify and recover IgG from biological media are of high-cost and time-consuming, resulting in high-cost products. In this sense, the search for cost-effective technologies to obtain highly pure and active IgG is highly required. The present work proposes a simple and efficient method for the purification and recovery of IgG from rabbit serum using magnetic iron oxide nanoparticles (magnetite, Fe3O4) coated with hybrid shells of a siliceous material modified with the anionic polysaccharide κ-carrageenan. Experimental parameters such as pH, contact time between the hybrid magnetic nanoparticles (HMNPs) and rabbit serum, and total protein concentration or dilution factor of serum were evaluated. The best results were achieved at pH 5.0, with a contact time of 60 min and using a rabbit serum with a total protein concentration of 4.8 mg·mL-1. Under these conditions, it was obtained an IgG purification factor and adsorption yield onto the HMNPs of 3.0 and 90%, respectively. The desorption of IgG from the HMNPs was evaluated using two strategies: a KCl aqueous solution and buffered aqueous solutions. Comparing to the initial rabbit serum, an IgG purification factor of 2.7 with a recovery yield of 74% were obtained using a buffered aqueous solution at pH 7.0. After desorption, the secondary structure of IgG and other proteins was evaluated by circular dichroism and no changes in the secondary structure were observed, meaning that the IgG integrity is kept after the adsorption and desorption steps. In summary, the application of HMNPs in the purification of IgG from serum samples has a high potential as a new downstream platform.

Effects of Amorphous Silica Nanopowders on the Avoidance Behavior of Five Soil Species-A Screening Study.

Silica nanoparticles (SiO2NPs) are one of the most used in commercial products and biomedical tools, however, their environmental effects have not been fully described. Although negative effects of SiO2NPs on the behavior of freshwater invertebrates have been reported, the knowledge is limited, especially the effect of nanopowders in terrestrial organisms. Accordingly, the aim of the present study is to understand the effects of SiO2NPs on the avoidance behavior of five soil species, whose niche may differ thus contributing to differential harmful SiO2NPs effects. Hence, avoidance assays testing SiO2NPs concentrations of 0, 10, 100, 250, 500 and 1000 mg/kg were performed with Enchytraeus crypticus, Folsomia candida, Tenebrio molitor, Porcellionides pruinosus and Eisenia fetida. SiO2NPs induced different behavioral effects, depending on the invertebrate ecology/habitat, exposure route and physiology. T. molitor, P. pruinosus and F. candida did not avoid contaminated soil; however, E. crypticus and E. fetida significantly avoided SiO2NPs spiked soil. Since these terrestrial worms (oligochaetes) live mostly burrowed in the soil, this can provide greater opportunity for SiO2NPs' uptake. On the other hand, the other tested organisms mainly living on the upper part of the soil did not avoid the SiO2NPs spiked soil. The avoidance data obtained here also highlight the need for further studies to understand whether (or not) the detected behavioral responses are linked to either neurotransmission processes or sensorial aspects of the biological models.

Design of Multifunctional Titania-Based Photocatalysts by Controlled Redox Reactions.

This work aims at the preparation of multifunctional titania-based photocatalysts with inherent capabilities for thermal co-activation and stabilisation of anatase polymorph, by designing the phase composition and microstructure of rutile-silicon carbide mixture. The processing involved a conventional solid state route, including partial pre-reduction of rutile by SiC in inert Ar atmosphere, followed by post-oxidation in air. The impacts of processing conditions on the phase composition and photocatalytic activity were evaluated using Taguchi planning. The XRD studies confirmed the presence of rutile/anatase mixtures in the post-oxidised samples. The results emphasise that pre-reduction and post-oxidation temperatures are critical in defining the phase composition, while post-oxidation time is relevant for the photocatalytic performance. Microstructural studies revealed the formation of core-shell particles, which can suppress the photocatalytic activity. The highest apparent reaction rate of the photodegradation of methylene blue was observed for the sample pre-reduced in Ar at 1300 °C for 5 h and then calcined in air at 400 °C for 25 h. Though its performance was ~1.6-times lower than that for the same amount of nanostructured industrial P25 photocatalyst, it was achieved in the material possessing 2-3 times lower surface area and containing ~50 mol% of SiO2 and SiC, thus demonstrating excellent prospects for further improvements.

Glycan affinity magnetic nanoplatforms for urinary glycobiomarkers discovery in bladder cancer.

Bladder Cancer (BC) presents one of the highest recurrence rates amongst solid tumours and constitutes the second deadliest disease of the genitourinary track. Non-invasive identification of patients facing disease recurrence and/or progression remains one of the most critical and challenging aspects in disease management. To contribute to this goal, we demonstrate the potential of glycan-affinity glycoproteomics nanoplatforms for urinary biomarkers discovery in bladder cancer. Briefly, magnetic nanoprobes (MNP) coated with three broad-spectrum lectins, namely Concanavalin A (ConA; MNP@ConA), Wheat Germ Agglutinin (WGA; MNP@WGA), and Sambucus nigra (SNA; MNP@SNA), were used to selectively capture glycoproteins from the urine of low-grade and high-grade non-muscle invasive as well as muscle-invasive BC patients. Proteins were identified by nano-LC MALDI-TOF/TOF and data was curated using bioinformatics tools (UniProt, NetOGlyc, NetNGlyc, ClueGO app for Cytoscape and Oncomine) to highlight clinically relevant species. Accordingly, 63 glycoproteins were exclusively identified in cancer samples compared with healthy controls matching in age and gender. Specific glycoprotein sets exclusively found in low-grade non-muscle invasive bladder tumours may aid early diagnosis, while those only found in high-grade non-invasive and muscle-invasive tumours hold potential for accessing progression. Amongst these proteins is bladder cancer stem-cell marker CD44, which has been associated with poor prognosis. Orthogonal validation studies by slot-blotting demonstrated an elevation in urine CD44 levels of high-grade patients, which became more pronounced upon muscle-invasion, in mimicry of the primary tumour. These observations demonstrate the potential of MNP@lectins for identification of clinically relevant glycoproteomics signatures in bladder cancer. Future clinical validation in a larger and well characterized patient subset is required envisaging clinical translation of the results.

Glycoprotein enrichment method using a selective magnetic nano-probe platform (MNP) functionalized with lectins.

Protein post-translational modifications (PTMs) have increasingly become a research field of incredible importance to fully understand the regulation of biological processes in health and disease. Among PTMs, glycosylation is one of the most studied for which contributed the development and improvement of enrichment techniques. Nowadays, glycoprotein enrichment methods are based on lectin affinity, covalent interactions, and hydrophilic interaction liquid chromatography (HILIC). Nonetheless, the nanotechnology era has fetched new methods to enrich glycoproteins from complex samples as human biological fluids. For instance, magnetic nanoparticles (MNPs) are being used as an interesting enrichment approach allowing a better characterization of glycoproteins and glycopeptides.In this chapter, we describe an enrichment method based on MNPs functionalized with lectins (Concavalin A, wheat germ agglutinin, and Maackia amurensis lectin) to enrich specific sets of glycoproteins from biological fluids. Moreover, it is proposed a bioinformatic strategy to deal with data retrieved from mass spectrometry analysis of enriched samples aiming the identification of relevant biological processes modulated by a given stimuli and, ultimately, of new biomarkers for disease screening/management.

Bionanoconjugation for proteomics applications - An overview.

Formed as an interdisciplinary domain on the basis of Human Genome Project, Proteomics aims at the large-scale study of proteins. The enthusiasm that resulted from obtaining the complete human genetic information has, however, been chastened by the realization that this information contributes little to the comprehension and knowledge of the expressed proteins. In the wake of this realization, the Human Proteome Project (HUPO) was founded, which is a global, collaborative initiative, aiming at the complete characterization of the proteins of all protein-coding genes. Nonetheless, the rapid detection of these molecules in complex biological samples under conditions considered to be of clinical relevance is extremely difficult, requiring the development of very sensitive, robust, reproducible and high throughput platforms. Nanoproteomics has emerged as a feasible, promising option, offering short assay times, low sample consumption, ultralow detection and high throughput capacity. Additionally, the successful synthesis of biomolecules and nanoparticle hybrids yields systems which often exhibit new or improved features. Herein, we overview the recent advances in bioconjugation at the nanolevel and, specifically, their application in Proteomics, discussing not only the merits and prospects of Proteomics, but also present day limitations.

Synthesis and optimization of lectin functionalized nanoprobes for the selective recovery of glycoproteins from human body fluids.

Biomedical sciences, and in particular biomarker research, demand efficient glycoprotein enrichment platforms. Herein magnetic nanoprobes (MNP), after being coated with three broad-spectrum lectins-concanavalin A (ConA), wheat germ agglutinin (WGA), and Maackia amurensis lectin (MA)-were utilized to selectively capture glycoproteins from human body fluids. Additionally, a new methodology, based on protection of the lectins with their target sugars prior to coupling with MNPs, was proposed to overcome the nonspecific nature of conjugation. This approach contributed to preserve lectin conformation, increasing by 40% and 90% the affinity of ConA and MA for glycoproteins in relation to synthesis with nonprotected lectins. Optimal operating conditions (temperature, time) and maximum binding capacities were further determined for each lectin by use of fetuin as a reference. The enhanced performance of lectin-based nanoplatforms was demonstrated by comparing MNP@ConA with conventional Sepharose@ConA. These experiments have shown that ConA immobilized on MNP exhibited 5 times higher affinity for fetuin and ovalbumin when compared with Sepharose@ConA with the same amount of immobilized lectin. MNP@Lectins were then applied to human serum, saliva, and urine and the recovered proteins were digested with trypsin and analyzed by nano-HPLC MALDI-TOF/TOF. This allowed the identification of 180 proteins, 90% of which were found to be glycosylated by use of bioinformatics tools, therefore revealing low levels of unspecific binding. Thus, MNP@lectins have proved to be a valuable tool for glycoproteomic studies, particularly when dealing with minute amounts of material.