Graphene-based immunoassay for human lipocalin-2

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1 pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4 °C in 0.1 M phosphate-buffered saline (PBS) for 8 weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings.     

Development of glutathione-coupled cantilever for the single-molecule force measurement by scanning force microscopy

The accuracy and the fidelity of a single-molecule force measurement largely rely on how the molecule of interest is attached to the solid substrate surface (bead, cantilever, cover glass and etc.). A site-specific attachment of a protein without affecting its structure and enzymatic function has been a major concern. Here, we established a glutathione-coupled cantilever to which any glutathione S-transferase (GST)-fused proteins can be attached in a desired direction. The rupture force between glutathione and GST was approximately 100 pN on average. By using this cantilever, we succeeded in measuring the interaction force between importin alpha and importin beta.     

Beta-lactoglobulin assembles into amyloid through sequential aggregated intermediates

We have investigated the aggregation and amyloid fibril formation of bovine β-lactoglobulin variant A, with a focus on the early stages of aggregation. We used noncovalent labeling with thioflavin T and 1-anilino-8-naphthalenesulfonate to follow the conformational changes occurring in β-lactoglobulin during aggregation using time resolved luminescence. 1-Anilino-8-naphthalenesulfonate monitored the involvement of the hydrophobic core/calyx of β-lactoglobulin in the aggregation process. Thioflavin T luminescence monitored the formation of amyloid. The luminescence lifetime distributions of both probes showed changes that could be attributed to conformational changes occurring during and following aggregation. To correlate the luminescence measurements with the degree of aggregation and the morphology of the aggregates, we also measured dynamic light scattering and atomic force microscopy images. We evaluated the relative stability of the intermediates with an assay that is sensitive to aggregation reversibility. Our results suggest that initial aggregation during the first 5 days occurred with partial disruption of the characteristic calyx in β-lactoglobulin. As the globular aggregates grew from days 5 to 16, the calyx was completely disrupted and the globular aggregates became more stable. After this second phase of aggregation, conversion into a fibrillar form occurred, marking the growth phase, and still more changes in the luminescence signals were observed. Based on these observations, we propose a three-step process by which monomer is converted first into weakly associated aggregates, which rearrange into stable aggregates, which eventually convert into protofibrils that elongate in the growth phase.     

Enantiomeric separation of β‐blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

The separation methods of the enantiomers of two β‐blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non‐covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β‐blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10 minutes under the optimized conditions: 30 mM phosphate buffer (pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.     

Easy parallel screening of reagent stability,quality control,and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1‐{[1‐(Cyano‐2‐ethoxy‐2‐oxo‐ethylidenaminooxy)dimethylamino‐morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma‐Aldrich, St. Louis, MO, USA) by SPPS of Leu‐Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3‐aminopropyl)triethyoxysilane‐modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Multi-stage delivery nano-particle systems for therapeutic applications

Background

The daunting task for drug molecules to reach pathological lesions has fueled rapid advances in Nanomedicine. The progressive evolution of nanovectors has led to the development of multi-stage delivery systems aimed at overcoming the numerous obstacles encountered by nanovectors on their journey to the target site.

Scope of review

This review summarizes major findings with respect to silicon-based drug delivery vectors for cancer therapeutics and imaging. Based on rational design, well-established silicon technologies have been adapted for the fabrication of nanovectors with specific shapes, sizes, and porosities. These vectors are part of a multi-stage delivery system that contains multiple nano-components, each designed to achieve a specific task with the common goal of site-directed delivery of therapeutics.

Major conclusions

Quasi-hemispherical and discoidal silicon microparticles are superior to spherical particles with respect to margination in the blood, with particles of different shapes and sizes having unique distributions in vivo. Cellular adhesion and internalization of silicon microparticles is influenced by microparticle shape and surface charge, with the latter dictating binding of serum opsonins. Based on in vitro cell studies, the internalization of porous silicon microparticles by endothelial cells and macrophages is compatible with cellular morphology, intracellular trafficking, mitosis, cell cycle progression, cytokine release, and cell viability. In vivo studies support superior therapeutic efficacy of liposomal encapsulated siRNA when delivered in multi-stage systems compared to free nanoparticles.This article is part of a Special Issue entitled Nanotechnologies - Emerging Applications in Biomedicine.     

Anthraquinones quinizarin and danthron unwind negatively supercoiled DNA and lengthen linear DNA

The intercalating drugs possess a planar aromatic chromophore unit by which they insert between DNA bases causing the distortion of classical B-DNA form. The planar tricyclic structure of anthraquinones belongs to the group of chromophore units and enables anthraquinones to bind to DNA by intercalating mode. The interactions of simple derivatives of anthraquinone, quinizarin (1,4-dihydroxyanthraquinone) and danthron (1,8-dihydroxyanthraquinone), with negatively supercoiled and linear DNA were investigated using a combination of the electrophoretic methods, fluorescence spectrophotometry and single molecule technique an atomic force microscopy. The detection of the topological change of negatively supercoiled plasmid DNA, unwinding of negatively supercoiled DNA, corresponding to appearance of DNA topoisomers with the low superhelicity and an increase of the contour length of linear DNA in the presence of quinizarin and danthron indicate the binding of both anthraquinones to DNA by intercalating mode.     

An atomic force microscopy method for the detection of binding forces between bacteria and a lipid bilayer containing higher order gangliosides

We developed an atomic force microscopy (AFM) method to determine the binding forces between a model cell wall plasma membrane and Vibrio cholerae. V. cholerae cells were covalently attached to AFM probes and forces were determined against a lipid bilayer containing sialic acid (N-acetylneuraminic acid) molecules as well as several control surfaces.     

Nanopore sensor for fast label-free detection of short double-stranded DNAs

Functionalizing surface enhanced the molecular sensing ability of a fabricated nanopore by increasing the translocation duration time for a short double-stranded DNA. The surface of nanopore was derivatized with γ-aminopropyltriethoxysilane and the positively charged surface attracted DNA molecules when they were in the vicinity of nanopore. The translocation duration time of DNA increased due to the strong electrostatic interaction and it enabled us to detect a short double-stranded DNA (<1 kbp) that is under the size limit of a conventional solid state nanopore sensor. Both 539 and 910 bp double-stranded DNAs were analyzed with the surface functionalized nanopore and their translocation kinetics are presented in this work. The new feature of the surface modified nanopore that can detect short double-stranded DNA molecules could readily be applied for a rapid label-free diagnostic analysis in a Lab-On-a-Chip type DNA sensor.     

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.