Disposable biosensor based on graphene oxide conjugated with tyrosinase assembled gold nanoparticles

A highly efficient enzyme-based screen printed electrode (SPE) was obtained by using covalent attachment between 1-pyrenebutanoic acid, succinimidyl ester (PASE) adsorbing on the graphene oxide (GO) sheets and amines of tyrosinase-protected gold nanoparticles (Tyr-Au). Herein, the bi-functional molecule PASE was assembled onto GO sheets. Subsequently, the Tyr-Au was immobilized on the PASE-GO sheets forming a biocompatible nanocomposite, which was further coated onto the working electrode surface of the SPE. The characterization of obtained nanocomposite and modified SPE surface was investigated by atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM). Attributing to the synergistic effect of GO-Au integration and the good biocompatibility of the hybrid-material, the fabricated disposable biosensor (Tyr-Au/PASE-GO/SPE) exhibited a rapid amperometric response (less than 6s) with a high sensitivity and good storage stability for monitoring catechol. This method shows a good linearity in the range from 8.3×10(-8) to 2.3×10(-5) M for catechol with a squared correlation coefficient of 0.9980, a quantitation limit of 8.2×10(-8) M (S/N=10) and a detection limit of 2.4×10(-8) M (S/N=3). The Michaelis-Menten constant was measured to be 0.027 mM. This disposable tyrosinase biosensor could offer a great potential for rapid, cost-effective and on-field analysis of phenolic compounds.     

Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers

Background  

Bio-conjugated nanoparticles are important analytical tools with emerging biological and medical applications. In this context, in situ conjugation of nanoparticles with biomolecules via laser ablation in an aqueous media is a highly promising one-step method for the production of functional nanoparticles resulting in highly efficient conjugation. Increased yields are required, particularly considering the conjugation of cost-intensive biomolecules like RNA aptamers.     

Synthesis of intracellular and extracellular gold nanoparticles with a green machine and its antifungal activity

Green synthesis method is being increasingly used in the development of safe, stable, and eco-friendly nanostructures with biological resources. In this study, extracellular and intracellular synthesis of gold nanoparticles (AuNPs) was carried out using green algae Chlorella sorokiniana Shihira & R.W. Fresh algae were isolated and identified from Musaözü Pond located in the province of Eskişehir and then extraction process were performed. Optimization studies were studied using pH value, metal salt concentration, and time parameters for extracellular synthesis and using only time parameter for intrasellular synthesis. Since more controlled and optimum conditions can be achieved in the production of AuNPs by extracellular synthesis, these nanoparticles (NPs) were used for characterization and antifungal activity studies. Optical, physical, and chemical properties of synthesized NPs were characterized by UV visible spectrophotometer (UV-Vis), dynamic light scattering (DLS), Zetasizer, X-Ray diffraction (XRD), Fourier transform ınfrared spectroscopy (FTIR), field emission scanning electron microscope (FE-SEM), ınductively coupled plasma mass spectrometer (ICP-MS) and transmission electron microscope (TEM) analysis. The optimum conditions for AuNPs synthesis were determined as 1 mM for HauCl₄ concentration, 6 for pH value, and 60th min for time. AuNPs obtained from extracellular synthesis from C. sorokiniana extract are 5–15 nm in size and spherical shape. TEM images of extracellular synthesis show noticeable cell wall and membrane damages, cytoplasma dissolutions, and irregularities. AuNPs obtained by intracellular synthesis are in 20–40 nm size and localized in the cell wall and cytoplasm. These NPs exhibited significant antifungal activity against C. tropicalis, C. glabrata, and C. albicans isolates. AuNPs obtained by algae-mediated green synthesis have a significant potential for medical and industrial use, and this eco-friendly synthesis method can be easily scaled for future studies.     

Structure and activity of apoferritin-stabilized gold nanoparticles

A simple method for synthesizing gold nanoparticles stabilized by horse spleen apoferritin (HSAF) is reported using NaBH(4) or 3-(N-morpholino)propanesulfonic acid (MOPS) as the reducing agent. AuCl(4)(-) reduction by NaBH(4) was complete within a few seconds, whereas reduction by MOPS was much slower; in all cases, protein was required during reduction to keep the gold particles in aqueous solution. Transmission electron microscopy (TEM) showed that the gold nanoparticles were associated with the outer surface of the protein. The average particle diameters were 3.6 and 15.4 nm for NaBH(4)-reduced and MOPS-reduced Au-HSAF, respectively. A 5-nm difference in the UV-Vis absorption maximum was observed for NaBH(4)-reduced (530 nm) and MOPS-reduced Au-HSAF (535 nm), which was attributed to the greater size and aggregation of the MOPS-reduced gold sample. NaBH(4)-reduced Au-HSAF was much more effective than MOPS-reduced Au-HSAF in catalyzing the reduction of 4-nitrophenol by NaBH(4), based on the greater accessibility of the NaBH(4)-reduced gold particle to the substrate. Rapid reduction of AuCl(4)(-) by NaBH(4) was determined to result in less surface passivation by the protein. Methods for studying ferritin-gold nanoparticle assemblies may be readily applied to other protein-metal colloid systems.     

Synthesis and DNA-cleaving activity of lactenediynes conjugated with DNA-complexing moieties

Lactenediynes are compounds characterized by the fusion of a beta-lactam with a cyclodeca-3-ene-1,5-diyne. In this work the most promising members of this family have been activated by attaching a carbalkoxy or a carbamoyl group to the azetidinone nitrogen, and conjugated to various DNA-complexing moieties, either acting by intercalation or through groove binding. These conjugated artificial enediynes have been demonstrated to possess in vitro ability to produce single and double strand cleavage of plasmid DNA. As potency and capacity to induce double cut, they rank among the best simple enediyne analogues ever prepared. A thorough investigation was carried out in order to develop the best suited linkers for assembling these conjugates.     

Magnetic single-enzyme nanoparticles with high activity and stability

Magnetic single-enzyme nanoparticles (SENs) encapsulated within a composite inorganic/organic polymer network were fabricated via the surface modification and in situ aqueous polymerization of separate enzyme molecule. The resultant nanoparticles were characterized by transmission electron microscope (TEM), Fourier transform infrared (FTIR) spectrometer and X-ray diffraction (XRD). These particles are almost spherical in shape and have a unique size of about 50 nm in diameter. Electrical and magnetic measurements reveal that the magnetic SENs have a conductivity of 2.7 × 10⁻³ S cm⁻¹, and are superparamagnetic with a saturation magnetization of 14.5 emu g⁻¹ and a coercive force of 60 Oe. Compared with free enzyme, encapsulated enzyme exhibits a strong tolerance to the variation of solution pH, high temperature, organic solvent and long-term storage, thus showing significantly enhanced enzyme performance and stability.     

The in vitro inhibition effect of 2 nm gold nanoparticles on non-enzymatic glycation of human serum albumin

The reaction of amino groups of protein and the carbonyl groups of reducing sugar molecules, non-enzymatically induce a series of chemical reactions that form a heterogeneous group of compounds known as advanced glycation end products (AGEs). The accumulation of AGEs is associated with various disease conditions that include complications in diabetes, Alzheimer's disease and aging. The current study monitored the extent of non-enzymatic glycation of human serum albumin (HSA) in order to estimate the formation of HSA related AGEs in the presence of 2 nm gold nanoparticles. The rate of glycation was evaluated using several analytical methods. Physiological concentrations of HSA and glyceraldehyde mixtures, incubated with various concentrations of negatively charged 2 nm gold nanoparticles, resulted in a lower reaction rate than mixtures without 2GNP. Moreover, increasing concentrations of gold nanoparticles exhibited a pronounced reduction in AGE formation. High performance liquid chromatography, UV-visible spectroscopy and circular dichroism analytical methods provide reliable techniques for evaluating AGE formation of HSA adducts.     

Synthesis and acetylcholinesterase/butyrylcholinesterase inhibition activity of new tacrine-like analogues

The synthesis and preliminary results for acetylcholinesterase and butyrylcholinesterase inhibition activity of a series of pyrano[2,3-b]quinolines (2, 3) and benzonaphthyridines (5, 6) derivatives are described. These molecules are tacrine-like analogues which have been prepared from readily available polyfunctionalized ethyl [6-amino-5-cyano-4H-pyrans and 6-amino-5-cyanopyridines]-3-carboxylates via Friedlander condensation with selected ketones. These compounds showed moderate acetylcholinesterase inhibition activity, the more potent (2e, 5b) being 6 times less active than tacrine. The butyrylcholinesterase activity of some of these molecules is also discussed.     

Synthesis and in vitro acetylcholinesterase and butyrylcholinesterase inhibitory potential of hydrazide based Schiff bases

To discover multifunctional agents for the treatment of Alzheimer’s disease, a series of hydrazide based Schiff bases were designed and synthesized based on multitarget-directed strategy. We have synthesized twenty-eight analogs of hydrazide based Schiff bases, characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase and butyrylcholinesterase inhibition. All compounds showed varied degree of acetylcholinesterase and butyrylcholinesterase inhibition when compared with standard Eserine. Among the series, compounds 10, 3 and 24 having IC₅₀ values 4.12 ± 0.01, 8.12 ± 0.01 and 8.41 ± 0.06 μM respectively showed potent acetylcholinesterase inhibition when compared with Eserine (IC₅₀ = 0.85 ± 0.0001 μM). Three compounds 13, 24 and 3 having IC₅₀ values 6.51 ± 0.01, 9.22 ± 0.07 and 37.82 ± 0.14 μM respectively showed potent butyrylcholinesterase inhibition by comparing with eserine (IC₅₀ = 0.04 ± 0.0001 μM). The remaining compounds also exhibited moderate to weak inhibitory potential. Structure activity relationship has been established. Through molecular docking studies the binding interaction was confirmed.     

A computational study of the deacylation mechanism of human butyrylcholinesterase

To investigate the mechanism of the deacylation reaction in the active site of human butyrylcholinesterase (BuChE), we carried out quantum mechanical (QM) calculations on cluster models of the active site built from a crystallographic structure. The models consisted of the substrate butyrate moiety, the catalytic triad of residues (Ser198, Glu325, and His438), the "oxy-anion hole" (Gly116, Gly117, and Ala199), the side chain of Glu197, four water molecules, the side chain of Ser225, and the peptide linkage between Val321 and Asn322. Analyses of the equilibrium geometries, electronic properties, and energies of the QM models gave insights into the catalytic mechanism. In addition, the QM calculations provided the data required to build a molecular mechanics representation of the reactive BuChE region that was employed in molecular dynamics simulations followed by molecular-mechanics-Poisson-Boltzmann (MM-PB) calculations. Subsequently, we combined the QM energies with average MM-PB energies to estimate the free energy of the reactive structures in the enzyme. The rate-determining step corresponds to the formation of a tetrahedral intermediate with a free-energy barrier of approximately 14.0 kcal/mol. The modulation of the BuChE activity, exerted by either neutral molecules (glycerol, GOL) or a second butyrylcholine (CHO) molecule bound to the cation-pi site, does not involve any significant allosteric effect. Interestingly, the presence of GOL or CHO stabilizes a product complex formed between a butyric acid molecule and BuChE. These results are in consonance with the crystallographic structure of BuChE, in which the catalytic Ser198 interacts with a butyric fragment, while the cation-pi site is occupied by one GOL molecule.     

Synthesis and in vitro evaluation of gold(I) thiosemicarbazone complexes for antimalarial activity

The reaction of thiosemicarbazones (TSCs) with [Au^I(THT)Cl], THT = tetrahydrothiophene, has been investigated. The resulting gold(I) complexes have been characterized by a range of spectroscopic techniques: NMR spectroscopy, mass spectrometry, microanalysis and infrared spectroscopy. The in vitro antimalarial data for gold(I) TSC complexes suggests that coordination of gold(I) to TSCs enhanced their efficacy against the malaria parasite Plasmodium falciparum and their inhibition of the parasite cysteine protease falcipain-2.     

Isolation of circulating human monocytes with high purity for proteomic analysis

We describe a simple method for isolation of human blood monocytes with the high purity (95-98%) required for proteomic analysis, which avoids contamination by other blood cells (platelets and lymphocytes) and the most abundant plasma proteins (albumin and immunoglobulins). Blood monocytes were purified by gradient centrifugation followed by positive selection with specific monoclonal antibodies coupled to paramagnetic beads. The elution conditions of the positive selection step were modified to avoid contamination with albumin. This method is compatible with flow cytometry which was used to assess the purity of the cell population. From 28 mL of blood, 10(7) monocytes with > 96% purity are routinely obtained. From the isolated monocytes 200-250 microg of protein could be recovered. The whole method can be performed in three hours. Similar results were obtained using a negative selection step but with lower purity (92%), increased cost and longer time. After solubilization of monocytes, the proteins were analyzed by two-dimensional gel electrophoresis (2-DE) in the 3-10, 4-7, 6-9 and 6-11 pH range. DNA was the main contaminant that interfered with the 2-DE and it was removed by treatment with DNAse. Image analysis of gels allowed the reproducible detection and quantification of 1500 spots in the 4-7 pH range and more than 2000 spots in total by combining (overlapping) 2-D gels in the 4-7, 6-9 and 6-11 pH range. This method is useful for clinical studies of monocytes from a large number of patients due to its rapidity and reproducibility, which permits comparative analysis of normal versus pathological samples and which allows follow up of the expressed proteins of monocytes from each patient.     

Recent advances in the antilung cancer activity of biosynthesized gold nanoparticles

Gold nanoparticles (Au-NPs) have been widely used in biomedical fields such as imaging, diagnosis, and treatment because of their special characteristics. Au-NPs can be synthesized using several methods, including the biological method, also called green or eco-friendly synthesis. Recent studies have reported the anticancer activity of biosynthesized Au-NPs, especially in lung cancer. This review focused on the advances in the antilung cancer activity of biosynthesized Au-NPs and its potential mechanisms.     

Influence of gold nanoparticles on activation of human blood neutrophils

Activation of neutrophils in the presence of gold nanoparticles is accompanied by formation of free-radical peroxidation products, recorded as a flash of chemiluminescence. The basis for the activation mechanism has its origins most likely in the influence of the gold particles on the membrane surface potential of neutrophils. Assessment of changes in the fluorescence intensity of the negatively charged ANS probe on the surface of model membranes upon adding different concentrations of gold nanoparticles indicates a change in the membrane surface charge density, which can cause cell activation.     

靶向金纳米棒对隐球菌活性影响的体外实验研究

目的：设计对隐球菌荚膜特异性标记的靶向金纳米棒,研究靶向金纳米棒的体外光热作用对隐球菌活性的影响。方法晶种生长法制备金纳米棒,偶联隐球菌荚膜抗体,检测表征,与隐球菌体外孵育,近红外激光照射,检测隐球菌活性变化。结果成功制备与荚膜抗体偶联的金纳米棒,体外近红外照射后,隐球菌活性较未偶联抗体的金纳米棒组显著降低。结论靶向性金纳米棒显著增强了近红外激光对隐球菌的光热效应,可用于治疗隐球菌感染的新尝试。     

Synthesis of chitosan-stabilized gold nanoparticles in the absence/presence of tripolyphosphate

Gold nanoparticles were prepared by reducing gold salt with a polysaccharide, chitosan, in the absence/presence of tripolyphosphate (TPP). Here, chitosan acted as a reducing/stabilizing agent. The obtained gold nanoparticles were characterized with UV--vis spectroscopy and transmission electron microscopy. The results indicated that the shape and size distribution of gold nanoparticles changed with the molecular weight and concentration of chitosan. More interestingly, the gelation of chitosan upon contacting with polyanion (TPP) can also affect the shape and size distribution of gold nanoparticles. By adding TPP to chitosan solution before the reduction of gold salt, gold nanoparticles have a bimodal size distribution, and at the same time, polygonal gold particles were obtained in addition to spherical gold nanoparticles.     

Synthesis and in vitro hydroxyapatite binding of peptides conjugated to calcium-binding moieties

To confer bone-binding properties to proteins and other biological agents that lack specific targeting capacity, model peptide-based molecules were synthesized containing poly(aspartic acid), poly(glutamic acid), or a bisphosphonate (pamidronate). These motifs have well-documented affinities to hydroxyapatite, a property desirable for the targeting of molecules to bone for drug delivery and tissue engineering applications. Model peptides of increasing molecular mass (5-33 amino acids) were directly conjugated to eight aspartic acids (Asp8), eight glutamic acids (Glu8), or pamidronate, purified by high-performance liquid chromatography, and characterized by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectroscopy. The modified peptides were incubated with hydroxyapatite in phosphate-buffered saline at physiological conditions over 24 h. This study revealed a significant amount (>90%) of conjugated peptides adsorbed to the hydroxyapatite as compared to unmodified peptides (<5%). It was found that while there were significant differences between the different hydroxyapatite-binding and control groups for all time points, the size of the peptide had no statistical effect on peptide-hydroxyapatite binding. These results demonstrate that bisphosphonate and oligopeptide conjugates hold great promise for the development of new bioactive molecules for bone-specific applications.