Optimization of antibody-conjugated magnetic nanoparticles for target preconcentration and immunoassays

Biosensors based on antibody recognition have a wide range of monitoring applications that apply to clinical, environmental, homeland security, and food problems. In an effort to improve the limit of detection of the Naval Research Laboratory (NRL) Array Biosensor, magnetic nanoparticles (MNPs) were designed and tested using a fluorescence-based array biosensor. The MNPs were coated with the fluorescently labeled protein, AlexaFluor647–chicken IgG (Alexa647–chick IgG). Antibody-labeled MNPs (Alexa647–chick–MNPs) were used to preconcentrate the target via magnetic separation and as the tracer to demonstrate binding to slides modified with anti-chicken IgG as a capture agent. A full optimization study of the antibody-modified MNPs and their use in the biosensor was performed. This investigation looked at the Alexa647–chick–MNP composition, MNP surface modifications, target preconcentration conditions, and the effect that magnetic extraction has on the Alexa647–chick–MNP binding with the array surface. The results demonstrate the impact of magnetic extraction using the MNPs labeled with fluorescent proteins both for target preconcentration and for subsequent integration into immunoassays performed under flow conditions for enhanced signal generation.     

A review on numerical modeling for magnetic nanoparticle hyperthermia: Progress and challenges

Recent progress in nanotechnology has advanced the development of magnetic nanoparticle (MNP) hyperthermia as a potential therapeutic platform for treating diseases. Due to the challenges in reliably predicting the spatiotemporal distribution of temperature in the living tissue during the therapy of MNP hyperthermia, critical for ensuring the safety as well as efficacy of the therapy, the development of effective and reliable numerical models is warranted. This article provides a comprehensive review on the various mathematical methods for determining specific loss power (SLP), a parameter used to quantify the heat generation capability of MNPs, as well as bio-heat models for predicting heat transfer phenomena and temperature distribution in living tissue upon the application of MNP hyperthermia. This article also discusses potential applications of the bio-heat models of MNP hyperthermia for therapeutic purposes, particularly for cancer treatment, along with their limitations that could be overcome.     

功能化磁性纳米粒子在固定化酶研究中的应用

酶是高效的生物催化剂,在生物技术领域有广泛的应用。然而,不可再生催化的高成本和酶的有效成分分离回收,是实现大规模工业化应用需要解决的关键问题。磁性纳米粒子(magnetic nanoparticles,MNPs)具有优异的磁回收性质。通过设计和制备功能化MNPs作为固定化酶的多功能载体,是解决这一问题的有效途径之一,可为酶的工业化大规模应用提供条件。近年来,功能化磁性纳米粒子在酶的固定化领域基于载体性质、固定化方法和应用有广泛研究。文中重点介绍了近年来各种功能化磁性纳米载体,特别是Fe3O4纳米粒子,在固定化酶中的应用。根据功能化试剂的差异分类,实例讨论了不同功能化修饰的磁性纳米载体对酶的固定化,包括硅烷修饰的磁性纳米载体、有机聚合物修饰的磁性纳米载体、介孔材料修饰的磁性纳米载体以及金属-有机骨架材料(metal-organic framework,MOF)修饰的磁性纳米载体。同时,结合可持续工业催化的发展要求,对磁性复合载体固定化酶的发展前景进行了展望。     

Promising insights into the kosmotropic effect of magnetic nanoparticles on proteins: The pivotal role of protein corona formation

Increasing concerns about biosafety of nanoparticles (NPs) has raised the need for detailed knowledge of NP interactions with biological molecules especially proteins. Herein, the concentration-dependent effect of magnetic NPs (MNPs) on bovine serum albumin and hen egg white lysozyme was explored. The X-ray diffraction patterns, zeta potential, and dynamic light scattering measurements together with scanning electron microscopy images were employed to characterize MNPs synthesized through coprecipitation method. Then, we studied the behavior of two model proteins with different surface charges and structural properties on interaction with Fe₃O₄. A thorough investigation of protein–MNP interaction by the help of intrinsic fluorescence at different experimental conditions revealed that affinity of proteins for MNPs is strongly affected by the similarity of protein and MNP surface charges. MNPs exerted structure-making kosmotropic effect on both proteins under a concentration threshold; however, binding strength was found to determine the extent of stabilizing effect as well as magnitude of the concentration threshold. Circular dichroism spectra showed that proteins with less resistance to conformational deformations are more prone to secondary structure changes upon adsorption on MNPs. By screening thermal aggregation of proteins in the presence of Fe₃O₄, it was also found that like chemical stability, thermal stability is influenced to a higher extent in more strongly bound proteins. Overall, this report not only provides an integrated picture of protein–MNP interaction but also sheds light on the molecular mechanism underling this process.     

A Noninvasive Method to Determine the Fate of Fe3O4 Nanoparticles following Intravenous Injection Using Scanning SQUID Biosusceptometry

Magnetic nanoparticles (MNPs) of Fe₃O₄ have been widely applied in many medical fields, but few studies have clearly shown the outcome of particles following intravenous injection. We performed a magnetic examination using scanning SQUID biosusceptometry (SSB). Based on the results of SSB analysis and those of established in vitro nonmagnetic bioassays, this study proposes a model of MNP metabolism consisting of an acute metabolic phase with an 8 h duration that is followed by a chronic metabolic phase that continues for 28 d following MNP injection. The major features included the delivery of the MNPs to the heart and other organs, the biodegradation of the MNPs in organs rich with macrophages, the excretion of iron metabolites in the urine, and the recovery of the iron load from the liver and the spleen. Increases in serum iron levels following MNP injection were accompanied by increases in the level of transferrin in the serum and the number of circulating red blood cells. Correlations between the in vivo and in vitro test results indicate the feasibility of using SSB examination for the measurement of MNP concentrations, implying future clinical applications of SSB for monitoring the hematological effects of MNP injection.     

In Vivo Screening of Hepatocellular Carcinoma Using AC Susceptibility of Anti-Alpha Fetoprotein-Activated Magnetic Nanoparticles

With antibody-mediated magnetic nanoparticles (MNPs) applied in cancer examinations, patients must pay at least twice for MNP reagents in immunomagnetic reduction (IMR) of in vitro screening and magnetic resonance imaging (MRI) of in vivo tests. This is because the high maintenance costs and complex analysis of MRI have limited the possibility of in vivo screening. Therefore, this study proposes novel methods for in vivo screening of tumors by examining the AC susceptibility of bound MNPs using scanning superconducting-quantum-interference-device (SQUID) biosusceptometry (SSB), thereby demonstrating high portability and improved economy. The favorable agreement between in vivo tests using SSB and MRI demonstrated the feasibility of in vivo screening using SSB for hepatocellular carcinoma (HCC) targeted by anti-alpha fetoprotein (AFP)-mediated MNPs. The magnetic labeling was also proved by in vitro tests using SSB and biopsy assays. Therefore, patients receiving bioprobe-mediated MNPs only once can undergo in vivo screening using SSB in the future.     

Synthesis,characterization and MRI application of dextran-coated Fe3O4 magnetic nanoparticles

Biocompatible ferrofluid based on dextran-coated Fe₃O₄ magnetic nanoparticles (MNPs) was prepared through a one-step method. In contrast to the conventional co-precipitation method, hydrazine hydrate was added as reducing agent and precipitator in the present investigation. The effects of hydrazine hydrate, the weight ratio of dextran to MNPs and the molecular weight of dextran on the dispersibility of MNPs in water were investigated. Also, the particles size of modified MNP and coating efficiency of dextran on MNPs were measured. In addition, biocompatible ferrofluid was intravenously injected into rabbits, the iron content in blood and organs at different times were measured by atomic absorption spectrometer, and the bio-distribution and the bio-transportation of ferrofluid in organs was examined. Then, the magnetic resonance (MR) images of liver, marrow and lymph were acquired by MRI experiments before and after intravenous injection of ferrofluid. Image analysis revealed that the MR signal intensity of these organs notably decreased after intensified by ferrofluid. However, when there existed tumors in organs, the signal intensity of tumor did not change after injection. From that the tumor can easily be identified, which indicated a potential application of the as-prepared MNP in functional molecular imaging for biomedical research and clinical diagnosis.     

Free Marine Natural Products Databases for Biotechnology and Bioengineering

Marine organisms and micro‐organisms are a source of natural compounds with unique chemical features. These chemical properties are useful for the discovery of new functions and applications of marine natural products (MNPs). To extensively exploit the potential implementations of MNPs, they are gathered in chemical databases that allow their study and screening for applications of biotechnological interest. However, the classification of MNPs is currently poor in generic chemical databases. The present availability of free‐access‐focused MNP databases is scarce and the molecular diversity of these databases is still very low when compared to the paid‐access ones. In this review paper, the current scenario of free‐access MNP databases is presented as well as the hindrances involved in their development, mainly compound dereplication. Examples and opportunities for using freely accessible MNP databases in several important areas of biotechnology are also assessed. The scope of this paper is, as well, to notify the latent potential of these information sources for the discovery and development of new MNPs in biotechnology, and push future efforts to develop a public domain MNP database freely available for the scientific community.     

Potential of Magnetic Nanofiber Scaffolds with Mechanical and Biological Properties Applicable for Bone Regeneration

Magnetic nanofibrous scaffolds of poly(caprolactone) (PCL) incorporating magnetic nanoparticles (MNP) were produced, and their effects on physico-chemical, mechanical and biological properties were extensively addressed to find efficacy for bone regeneration purpose. MNPs 12 nm in diameter were citrated and evenly distributed in PCL solutions up to 20% and then were electrospun into nonwoven nanofibrous webs. Incorporation of MNPs greatly improved the hydrophilicity of the nanofibers. Tensile mechanical properties of the nanofibers (tensile strength, yield strength, elastic modulus and elongation) were significantly enhanced with the addition of MNPs up to 15%. In particular, the tensile strength increase was as high as ∼25 MPa at 15% MNPs vs. ∼10 MPa in pure PCL. PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation of the nanofibers, with a weight loss of ∼20% in pure PCL, ∼45% in 10% MNPs and ∼60% in 20% MNPs. Apatite forming ability of the nanofibers tested in vitro in simulated body fluid confirmed the substantial improvement gained by the addition of MNPs. Osteoblastic cells favored the MNPs-incorporated nanofibers with significantly improved initial cell adhesion and subsequent penetration through the nanofibers, compared to pure PCL. Alkaline phosphatase activity and expression of genes associated with bone (collagen I, osteopontin and bone sialoprotein) were significantly up-regulated in cells cultured on PCL-MNP nanofibers than those on pure PCL. PCL-MNP nanofibers subcutaneously implanted in rats exhibited minimal adverse tissue reactions, while inducing substantial neoblood vessel formation, which however, greatly limited in pure PCL. In vivo study in radial segmental defects also signified the bone regeneration ability of the PCL-MNP nanofibrous scaffolds. The magnetic, bone-bioactive, mechanical, cellular and tissue attributes of MNP-incorporated PCL nanofibers make them promising candidate scaffolds for bone regeneration.     

Immobilization of recombinant pectate lyase from Clostridium thermocellum ATCC‐27405 on magnetic nanoparticles for bioscouring of cotton fabric

Recombinant pectate lyase from family 1 polysaccharide lyase (PL1B) was immobilized on synthesized magnetic nanoparticles (MNPs) after 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide hydrochloride activation. At 70 mg/mL MNPs 100% binding of 1 mg/mL PL1B was achieved. The immobilized PL1B‐MNP displayed activity of 20.3 and 18.2 U/mg against polygalacturonic acid and citrus pectin, respectively, which was higher than the activity of free PL1B, on the same substrates of 17.8 and 16.2 U/mg. The immobilized PL1B‐MNP showed 32 fold and 14 fold enhanced thermal stability at 80°C and 90°C, respectively as compared with free PL1B at same temperatures. At high temperature the immobilized PL1B‐MNP retained its activity for a longer duration than free PL1B. The immobilized PL1B‐MNP could be reused till five cycles and after that it retained 70% of initial activity. It could be easily recovered from the reaction mixture with the help of a magnet. Bioscouring of cotton fabric was carried out with immobilized PL1B‐MNP which showed efficient removal of pectin from the fabric surface. The enhanced wettability of fabric resulted in the decrease of the water absorbing time period from 3 min taken by the free PL1B treated fabric to 15 s taken by the immobilized PL1B‐MNP treated fabric. As per our knowledge this is the first attempt of bioscouring of coarse cotton fabric by pectinase immobilized on magnetic nanoparticles. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:231–244, 2017     

Preparation and fluorescence properties of fluorophore-labeled avidin–biotin system immobilized on Fe3O4 nanoparticles through functional indolequinone linker

We prepared and characterized a new class of fluorophore-labeled magnetic nanoparticles (MNPs) possessing a hypoxia-responsive unit to construct a hypoxia-selective emission system. The indolequinone derivative as a hypoxia-response unit bearing biotin was synthesized and immobilized on Fe₃O₄ MNP. Subsequent complexation of this functionalized MNP with fluorescein-labeled avidin formed fluorophore-labeled nanoparticles (AF-QB@MNP). The fluorescence intensity of AF-QB@MNP was suppressed because of the adjacent quenching function of the indolequinone moiety and MNP. Upon hypoxic treatment by NADPH:cytochrome P450 reductase, AF-QB@MNP was activated to liberate a fluorescence unit, leading to the significant enhancement of fluorescence emission, while a smaller enhancement in fluorescence emission occurred upon aerobic treatment. The AF-QB@MNP has a indispensable properties as a fluorescent probe for imaging of disease relevant hypoxic microenvironments.     

Compare Analysis for the Nanotoxicity Effects of Different Amounts of Endocytic Iron Oxide Nanoparticles at Single Cell Level

Developing methods that evaluate the cellular uptake of magnetic nanoparticles (MNPs) and nanotoxicity effects at single-cellular level are needed. In this study, magnetophoresis combining fluorescence based cytotoxicity assay was proposed to assess the viability and the single-cellular MNPs uptake simultaneously. Malignant cells (SKHep-1, HepG2, HeLa) were incubated with 10 nm anionic iron oxide nanoparticles. Prussian blue stain was performed to visualize the distribution of magnetic nanoparticles. MTT and fluorescence based assay analyzed the cytotoxicity effects of the bulk cell population and single cell, respectively. DAPI/PI stained was applied to evaluate death mechanism. The number of intracellular MNPs was found to be strongly correlated with the cell death. Significant differences between cellular MNP uptake in living and dead cells were observed. The method could be useful for future study of the nanotoxicity induced by MNPs.     

Maternal embryonic leucine zipper kinase (MELK) regulates multipotent neural progenitor proliferation

Maternal embryonic leucine zipper kinase (MELK) was previously identified in a screen for genes enriched in neural progenitors. Here, we demonstrate expression of MELK by progenitors in developing and adult brain and that MELK serves as a marker for self-renewing multipotent neural progenitors (MNPs) in cultures derived from the developing forebrain and in transgenic mice. Overexpression of MELK enhances (whereas knockdown diminishes) the ability to generate neurospheres from MNPs, indicating a function in self-renewal. MELK down-regulation disrupts the production of neurogenic MNP from glial fibrillary acidic protein (GFAP)-positive progenitors in vitro. MELK expression in MNP is cell cycle regulated and inhibition of MELK expression down-regulates the expression of B-myb, which is shown to also mediate MNP proliferation. These findings indicate that MELK is necessary for proliferation of embryonic and postnatal MNP and suggest that it regulates the transition from GFAP-expressing progenitors to rapid amplifying progenitors in the postnatal brain.     

A novel cell modification method used in biotransformation of glycerol to 3-HPA by Lactobacillus reuteri

The aim of the present study was to develop a new cell modification method to facilitate the cell separation from broth. In order to reduce the transfer limitation of substrate and product caused by general immobilization methods in the following biotransformation of glycerol, the carboxyl-functioned superparamagnetic nanoparticle (MNP) was directly attached to the surface of Lactobacillus reuteri for 3-hydroxypropionealdehyde producing. The modification process could be finished in several minutes by just adding MNP fluid into the bulk fermentation broth. The modified cells could be rapidly separated from the solution with the aid of magnetic field. The interaction between cell and MNP was shown by electron microscopy. The efficiency of the cells attached by MNPs for transformation of various concentrations of glycerol (100–400 mM) was studied at various temperatures (25–40 °C) and pH levels (5.8–7.5) with different cell concentrations (7.5–30 g/L). The 3- hydroxypropionealdehyde (HPA)/glycerol molar conversion under optimal condition (30 °C and pH 7) reached 70 %. The inactive modified cell could be reactivated easily by fresh medium and recovered the ability of glycerol conversion. MNPS distributing on cell surface had little adverse effect on cell activity. The modification method simplified the two-step production of 3-HPA by resting L. reuteri. The method of MNPs attached to cell surface is totally different from the traditional immobilization method in which the cell is attached to or entrapped in big carrier. The results obtained in this study showed that carboxyl-functioned MNP could be directly used as cell modification particle and realized cell recycle with the aid of magnetic field in bioprocess.     

Bacteria capture, lysate clearance, and plasmid DNA extraction using pH-sensitive multifunctional magnetic nanoparticles

A multifunctional magnetic nanoparticle (MNP)-assisted bioseparation method was developed to isolate plasmid DNA (pDNA) from Escherichia coli culture. Using the pH-sensitive carboxyl-modified magnetic nanoparticles, both cell capture and the subsequent removal of genomic DNA/protein complex after lysis can be achieved simply by magnetic separation. Furthermore, the yield and purity of pDNA extracted by MNPs are comparable to those obtained using organic solvents or commercial kits. This time- and cost-effective protocol does not require centrifugation or precipitation steps and has the potential for automated DNA extraction, especially within miniaturized lab chip applications.     

Magnetic fields suppress Pseudomonas aeruginosa biofilms and enhance ciprofloxacin activity

Due to the refractory nature of pathogenic microbial biofilms, innovative biofilm eradication strategies are constantly being sought. Thus, this study addresses a novel approach to eradicate Pseudomonas aeruginosa biofilms. Magnetic nanoparticles (MNP), ciprofloxacin (Cipro), and magnetic fields were systematically evaluated in vitro for their relative anti-biofilm contributions. Twenty-four-hour biofilms exposed to aerosolized MNPs, Cipro, or a combination of both, were assessed in the presence or absence of magnetic fields (Static one-sided, Static switched, Oscillating, Static + oscillating) using changes in bacterial metabolism, biofilm biomass, and biofilm imaging. The biofilms exposed to magnetic fields alone exhibited significant metabolic and biomass reductions (p < 0.05). When biofilms were treated with a MNP/Cipro combination, the most significant metabolic and biomass reductions were observed when exposed to static switched magnetic fields (p < 0.05). The exposure of P. aeruginosa biofilms to a static switched magnetic field alone, or co-administration with MNP/Cipro/MNP + Cipro appears to be a promising approach to eradicate biofilms of this bacterium.     

Multilayered Magnetic Nanoparticles as a Support in Solid-Phase Peptide Synthesis

The synthesis of multilayered magnetic nanoparticles (MNPs) for use as a support in solid-phase peptide synthesis (SPPS) is described. Silanization of magnetite (Fe₃O₄) nanoparticles with 3-(trimethoxysilyl)propyl methacrylate introduced polymerizable groups on the surface. Polymerization with allylamine, trimethylolpropane trimethacrylate, and trimethylolpropane ethoxylate (14/3 EO/OH) triacrylate provided a polymeric coating and amino groups to serve as starting points for the synthesis. After coupling of an internal reference amino acid and a cleavable linker, the coated MNPs were applied as the solid phase during synthesis of Leu-enkephalinamide and acyl carrier protein (65-74) by Fmoc chemistry. A “high-load” version of the MNP support (0.32 mmol/g) was prepared by four consecutive cycles of Fmoc-Lys(Fmoc)-OH coupling and Fmoc deprotection. Successful synthesis of Leu-enkephalin was demonstrated on the “high-load” MNPs. Chemical stability studies proved the particles to be stable under SPPS conditions and magnetization measurements showed that the magnetic properties of the particles were maintained throughout derivatizations and SPPS. The MNPs were further characterized by high-resolution transmission electron microscopy, inductively coupled plasma atomic emission spectrometry, elemental analysis, and nitrogen gas adsorption measurements.     

Global analysis of the perturbation effects of metal-based nanoparticles on soil nitrogen cycling

Although studies have investigated the effects of metal-based nanoparticles (MNPs) on soil biogeochemical processes, the results obtained thus far are highly variable. Moreover, we do not yet understand how the impact of MNPs is affected by experimental design and environmental conditions. Herein, we conducted a global analysis to synthesize the effects of MNPs on 17 variables associated with soil nitrogen (N) cycling from 62 studies. Our results showed that MNPs generally exerted inhibitory effects on N-cycling process rates, N-related enzyme activities, and microbial variables. The response of soil N cycling varied with MNP type, and exposure dose was the most decisive factor for the variations in the responses of N-cycling process rates and enzyme activities. Notably, Ag/Ag₂S and CuO had dose-dependent inhibitory effects on ammonia oxidation rates, while CuO and Zn/ZnO showed hormetic effects on nitrification and denitrification rates, respectively. Other experimental design factors (e.g., MNP size and exposure duration) also regulated the effect of MNPs on soil N cycling, and specific MNPs, such as Ag/Ag₂S, exerted stronger effects during long-term (>28 days) exposure. Environmental conditions, including soil pH, organic carbon, texture, and presence/absence of plants, significantly influenced MNP toxicity. For instance, the effects of Ag/Ag₂S on the ammonia oxidation rate and the activity of leucine aminopeptidase were more potent in acid (pH <6), organic matter-limited (organic carbon content ≤10 g kg⁻¹), and coarser soils. Overall, these results provide new insights into the general mechanisms by which MNPs alter soil N processes in different environments and underscore the urgent need to perform multivariate and long-term in situ trials in simulated natural environments.     

Statistical optimization of <Emphasis Type="Italic">Bacillus alcalophilus</Emphasis> α-amylase immobilization on iron-oxide magnetic nanoparticles

We report the statistical optimization of the immobilization of alkaline α-amylase [E.C. 3.2.1.1] from Bacillus alcalophilus onto nano-sized supermagnetic ironoxide nanoparticles (MNPs) for augmenting the cost effective industrial application of MNP-bound α-amylase. Both Plackett-Burman factorial design and response surface methodology (RSM) were employed to screen the influence of different parameters and the central effect of response on the α-amylase-iron oxide MNP binding process. The high coefficient of determination (R2) and analysis of variance (ANOVA) of the quadratic model indicated the competence of the proposed model. The size of the MNPs was confirmed by X-ray diffraction and scanning electron microscope analyses in which Fourier transform infrared spectroscopy suggested immobilization of the enzyme on iron-oxide MNPs. A significant improvement (∼ 26-fold) in specific activity, thermal and storage stability, and reusability of α-amylase after binding with iron-oxide MNP reinforced the improved biotechnological potential of the α-amylase iron-oxide MNP bioconjugate compared to free α-amylase. These results open new avenues for applying this MNP immobilized enzyme in different industrial sectors, notably in the paper and brewing industries.     

The gut microbiota,a key to understanding the health implications of micro(nano)plastics and their biodegradation

The effects of plastic debris on the environment and plant, animal, and human health are a global challenge, with micro(nano)plastics (MNPs) being the main focus. MNPs are found so often in the food chain that they are provoking an increase in human intake. They have been detected in most categories of consumed foods, drinking water, and even human feces. Therefore, oral ingestion becomes the main source of exposure to MNPs, and the gastrointestinal tract, primarily the gut, constantly interacts with these small particles. The consequences of human exposure to MNPs remain unclear. However, current in vivo studies and in vitro gastrointestinal tract models have shown that MNPs of several types and sizes impact gut intestinal bacteria, affecting gut homeostasis. The typical microbiome signature of MNP ingestion is often associated with dysbiosis and loss of resilience, leads to frequent pathogen outbreaks, and local and systemic metabolic disorders. Moreover, the small micro- and nano-plastic particles found in animal tissues with accumulated evidence of microbial degradation of plastics/MNPs by bacteria and insect gut microbiota raise the issue of whether human gut bacteria make key contributions to the bio-transformation of ingested MNPs. Here, we discuss these issues and unveil the complex interplay between MNPs and the human gut microbiome. Therefore, the elucidation of the biological consequences of this interaction on both host and microbiota is undoubtedly challenging. It is expected that microbial biotechnology and microbiome research could help decipher the extent to which gut microorganisms diversify and MNP-determinant species, mechanisms, and enzymatic systems, as well as become important to understand our response to MNP exposure and provide background information to inspire future holistic studies.