[Show abstract][Hide abstract] ABSTRACT: The N-hydroxysuccinimide (NHS) ester moiety is one of the most widely used amine reactive groups for covalent conjugation of proteins/peptides to other functional targets. In this study, a cleave-analyze approach was developed to quantify NHS ester groups conjugated to silica-coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs). The fluorophore dansylcadaverine was attached to Fe3O4@SiO2 magnetic nanoparticles (MNPs) via reaction with NHS ester groups, and then released from the MNPs by cleavage of the disulfide bond in the linker between the fluorophore and the MNPs moiety. The fluorophore released from Fe3O4@SiO2 MNPs was fluorometrically measured, and the amount of fluorophore should be equivalent to the quantity of the NHS ester groups on the surface of Fe3O4@SiO2 MNPs that participated in the fluorophore conjugation reaction. Another sensitive and semiquantitative fluorescence microscopic test was also developed to confirm the presence of NHS ester groups on the surface of Fe3O4@SiO2 MNPs. Surface-conjugated NHS ester group measurements were primarily performed on Fe3O4@SiO2 MNPs of 100–150 nm in diameter and also on 20-nm nanoparticles of the same type but prepared by a different method. The efficiency of labeling native proteins by NHS ester-coated Fe3O4@SiO2 MNPs was explored in terms of maximizing the number of MNPs conjugated per BSA molecule or maximizing the number of BSA molecules conjugated per each nanoparticle. Maintaining the amount of fresh NHS ester moieties in the labeling reaction system was essential especially when maximizing the number of MNPs conjugated per protein molecule. The methodology demonstrated in this study can serve as a guide in labeling the exposed portions of proteins by bulky multivalent labeling reagents.
Journal of Nanoparticle Research 09/2015; 17(9). DOI:10.1007/s11051-015-3133-z · 2.18 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Covalent labeling of solvent exposed amino acid residues using chemical reagents/crosslinkers followed by mass spectrometric analysis can be used to determine the solvent accessible amino acids of a protein. A variety of chemical reagents containing cleavable bonds were developed to label abundantly found lysine residues on the surface of protein. To achieve efficient separation of labeled peptides prior to mass spectrometric analysis, magnetic nanoparticles can be decorated with amino acid reactive functional groups and utilized for quick recovery of labeled peptides.  In this work, iron oxide magnetic nanoparticles (Fe3O4 MNPs) were synthesized by thermal decomposition method and coated with silica (SiO2@Fe3O4 MNPs) by reverse micro emulsion approach. The Fe3O4 MNPs and SiO2@Fe3O4 MNPs were characterized by TEM and XRD. The SiO2@Fe3O4 MNPs were further coated with amine groups and conjugated to N-hydroxysuccinimidyl (NHS) ester groups via a cleavable ester bond. Fluorescence based qualitative analysis of ester linked NHS ester modified SiO2@Fe3O4 MNPs was performed to confirm the presence of NHS ester group. The active NHS ester sites on the surface of SiO2@Fe3O4 MNPs were determined by depletion approach and found to be 694 active sites per 1 mg of SiO2@Fe3O4 MNPs. Free amine groups of a small peptide, ACTH (4–11) were labeled by ester linked, NHS ester modified SiO2@Fe3O4 MNPs under physiological conditions. Superparamagnetic nature of SiO2@Fe3O4 MNPs allowed quick and efficient magnetic separation of labeled peptides from the solution. The ester bond was further cleaved to separate labeled peptides followed by mass spectrometric analysis. The ester linked, NHS ester modified SiO2@Fe3O4 MNPs introduced a mass shift of 115.09 Da on amine groups of ACTH (4–11), which was confirmed by mass spectrometry.
[Show abstract][Hide abstract] ABSTRACT: The use of human serum albumin nanoparticles (HSAPs) as a drug carrier system for cancer treatment has proven successful through current marketable clinical formulations. Despite this success, there is a current lack of multi-functional HSAPs which offer combinational therapies of more than one proven technique. Gold nanorods (AuNRs) have also shown medicinal promise due to their photothermal therapy capabilities. In this study, a desolvation and crosslinking approach was employed to successfully encapsulate gold nanorods into HSAPs simultaneously with the chemotherapeutic drug paclitaxel (PAC); forming PAC-AuNR-HSAPs with desirable overall particle sizes of 299 ± 6 nm. The loading efficiency of paclitaxel into PAC-AuNR-HSAPs reached up to 3μg PAC/mg HSA. The PAC-AuNR-HSAPs experienced photothermal heating; with the bulk particle solution reaching up to 46 ˚C after 15 minutes of near-IR laser exposure. This heat increase marked the successful attainment of the temperature necessary to cause severe cellular hyperthermia and necrosis. The encasement strategy facilitated a colloidal hybrid treatment system capable of enhanced permeability and retention effects, photothermal ablation of cancer cells, and release of the active paclitaxel of up to 188 ng (from PAC-AuNR-HSAPs created with 30 mg HSA) in a single 15 minute irradiation session. When treated with PAC-AuNR-HSAPs containing 20 µg PAC/mL particle solution, 4T1 mouse breast cancer cells experienced ~ 82% cell death without irradiation and ~ 94% cell death after just one irradiation session. The results for PAC-AuNR-HSAPs were better than that of free PAC, which only killed ~ 77% of the cells without irradiation and ~ 80% with irradiation. The hybrid particle system also lends itself to future customizable external functionalities via conjugated targeting ligands such as antibodies. Internal entrapment of patient tailored medication combinations are also possible with this combination treatment platform, which may result in improved quality of life for those undergoing treatment.
[Show abstract][Hide abstract] ABSTRACT: Low density lipoprotein (LDL) is a major cholesterol carrier in human blood. Oxidations of apolipoprotein B-100 (apo B-100, LDL protein) could be pro-atherogenic and play critical roles in early stages of plaque formation in the arterial wall. The structure of apo B-100 is still poorly understood, partially due to its size (550 KDa, 4563 amino acids). To gain an insight into LDL structure, we mapped the regions of apo B-100 in human LDL which were prone to oxidation using peroxynitrite and hypochlorite as probes. In this study, LDL was incubated with various concentrations of peroxynitrite and sodium hypochlorite in bicarbonate buffer. The LDL protein apo B-100 was delipidated, denatured, alkylated and subjected to tryptic digestion. Tryptic peptides were analyzed employing liquid chromatography – tandem mass spectrometry (LC-MS/MS). Database search was performed against the apo B-100 database (P04114) using “SEQUEST” algorithm to identify peroxynitrite and hypochlorite mediated oxidations markers nitrotyrosine, nitrotryptophan, hydroxy-tryptophan and 3-chlorotyrosine. Several site specific oxidations were identified in apo B-100 after treatment of intact LDL particles with the oxidants. We hypothesize that these regions could be accessible to oxidant and critical for early events in atherosclerotic plaque deposition.This article is protected by copyright. All rights reserved
[Show abstract][Hide abstract] ABSTRACT: Abstract Progress has been made in using human serum albumin nanoparticles (HSAPs) as promising colloidal carrier systems for early detection and targeted treatment of cancer and other diseases. Despite this success, there is a current lack of multi-functional HSAP hybrids that offer combinational therapies. The size of the HSAPs has crucial importance on drug loading and in vivo performance and has previously been controlled via manipulation of pH and cross-linking parameters. Gold nanomaterials have also gained attention for medicinal use due to their ability to absorb near-infrared light, thus offering photothermal capabilities. In this study, the desolvation and cross-linking approach was employed to encapsulate gold nanorods, nanoparticles, and nanoshells into HSAPs. Incorporation of gold nanomaterials caused some changes in HSAP sizes, but the general size trends remained. This encasement strategy facilitated size-controlled HSAPs, in the range of 100-300 nm, loaded with gold nanostructures; providing composite particles which incorporate photothermally active components.
Journal of Microencapsulation 08/2014; 31(8):1-8. DOI:10.3109/02652048.2014.940012 · 1.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In order to assess the impact of sunlight on oil fate, Macondo Well Oil from the Deepwater Horizon (DWH) rig was mixed with pure water and irradiated with simulated sunlight. After irradiation, the water-soluble organics (WSO) from the dark and irradiated samples were extracted and characterized by ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Liquid-liquid extraction yielded two fractions from dark and irradiated water/oil mixtures: acidic WSOs (negative-ion electrospray (ESI)), and base/neutral WSOs (positive-ion ESI) coupled to FT-ICR MS to catalogue molecular-level transformations that occur to Macondo-derived WSOs after solar irradiation. Such direct measure of oil phototransformation has not been previously reported. The most abundant heteroatom class detected in the irradiated WSO acid fractions correspond to molecules that contain five oxygens (O5), while the most abundant acids in the dark samples contain two oxygen atoms per molecule (O2). Higher-order oxygen classes (O5 – O9) were abundant in the irradiated samples, but < 1.5 % relative abundance in the dark sample. The increased abundance of higher-order oxygen classes in the irradiated samples relative to the dark samples indicates that photooxidized components of the Macondo crude oil become water-soluble after irradiation. The base/neutral fraction showed decreased abundance of pyridinic nitrogen (N1) concurrent with an increased abundance of N1Ox classes after irradiation. The predominance of higher-order oxygen classes indicates that multiple photochemical pathways exist that result in oxidation of petroleum compounds.
[Show abstract][Hide abstract] ABSTRACT: Photochemical production of singlet oxygen from thin oil films over seawater and pure water was measured with furfuryl alcohol as a selective chemical probe. Oil was collected from the surface of the Gulf of Mexico following the Deepwater Horizon spill and from other sources. The loss of furfuryl alcohol and the formation of 6-hydroxy(2H)pyran-3(6H)-one were monitored. Total singlet oxygen formation was studied using high furfuryl alcohol concentrations and varying exposure time. The total amount of singlet oxygen produced in 1 hour irradiations of thin oil films (100 mg, 60 microns thick) over Gulf of Mexico water and pure water were 1.9 ± 0.4 × 10-5 and 1.6 ± 0.3 × 10-5 mol, respectively. After initial tests were performed, titanium dioxide (TiO2) nanomaterials were added to the system in two different concentrations to study the effects of singlet oxygen formation in the presence of a photocatalyst. The addition of TiO2 nanoparticles did not significantly change the observed formation rate of singlet oxygen. Steady state concentrations of photoproduced singlet oxygen were also determined and found to be near 1 × 10-12 M in water under thin films of oil, which is considerably greater than values previously observed for pure seawater. This study illustrates that oil is a source of singlet oxygen when exposed to sunlight. The fate of oil and other dissolved species will be heavily dependent on the formation and reaction of singlet oxygen in thin oil films on water.
Journal of Photochemistry and Photobiology A Chemistry 07/2014; 286. DOI:10.1016/j.jphotochem.2014.04.016 · 2.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sunlight exposed oil films on seawater or pure water produced substantial amounts of hydroxyl radical as a result of irradiation. Oil was collected from the surface of the Gulf of Mexico following the Deepwater Horizon spill and exposed to simulated sunlight in thin films over water. Photochemical production of hydroxyl radical was measured with benzoic acid as a selective chemical probe in the aqueous layer. Total hydroxyl radical formation was studied using high benzoic acid concentrations and varying exposure time. The total amount of hydroxyl radical produced in 24h irradiations of thin oil films over Gulf of Mexico water and pure water were 3.7×10(-7) and 4.2×10(-7)moles respectively. Steady state concentrations of hydroxyl radical were measured using a competition kinetics approach. Hydroxyl radical concentrations of 1.2×10(-16) to 2.4×10(-16)M were observed for seawater and pure water under oil films. Titanium dioxide (TiO2) nanomaterials were added to the system in an effort to determine if the photocatalyst would enhance oil photodegradation. The addition of TiO2 nanoparticles dramatically changed the observed formation rate of hydroxyl radical in the systems with NP water at pH 3, showing increased formation rate in many cases. With photocatalyst, the steady state concentration of radical decreased, predominantly due to an increase in the hydroxyl radical scavenging rate with oxide present. This study illustrates that oil is a strong and important source of hydroxyl radical when exposed to sunlight. The fate of oil and other dissolved species following oil spills will be heavily dependent on the formation and fate of hydroxyl radical.
[Show abstract][Hide abstract] ABSTRACT: We report a facile way to fabricate three-dimensional (3D) Ni–TiO2 core–shell nanowire arrays through anodic aluminum oxide template-assisted sol–gel TiO2 nanotube shell growth followed by Ni core using room temperature constant current electrodeposition. The 3D Ni–TiO2 nanowire-based dye-sensitized solar cell (DSSC) endows a 67% increase in conversion efficiency as compared with the TiO2 nanotube DSSC and maximum conversion efficiency of 5.07% was obtained by surface treating the photoanode with TiCl4, which provides enhanced light scattering and surface passivation. Indeed, this work paves the way to build reliable 3D Ni–TiO2 nanostructured photoanodes for highly efficient DSSCs.
[Show abstract][Hide abstract] ABSTRACT: In this paper we describe the fabrication and characterization of new liposome encapsulated quantum dot-fluorescence resonance energy transfer (FRET)-based probes for monitoring the enzymatic activity of phospholipase A2. To fabricate the probes, luminescent CdSe/ZnS quantum dots capped with trioctylphosphine oxide (TOPO) ligands were incorporated into the lipid bilayer of unilamellar liposomes with an average diameter of approximately 100 nm. Incorporating TOPO capped quantum dots in liposomes enabled their use in aqueous solution while maintaining their hydrophobicity and excellent photophysical properties. The phospholipid bilayer was labeled with the fluorophore NBD C6-HPC (2-(6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexa decanoyl-sn-glycero-3-phosphocholine). The luminescent quantum dots acted as FRET donors and the NBD dye molecules acted as FRET acceptors. The probe response was based on FRET interactions between the quantum dots and the NBD dye molecules. The NBD dye molecules were cleaved and released to the solution in the presence of the enzyme phospholipase A2. This led to an increase of the luminescence of the quantum dots and to a corresponding decrease in the fluorescence of the NBD molecules, because of a decrease in FRET efficiency between the quantum dots and the NBD dye molecules. Because the quantum dots were not attached covalently to the phospholipids, they did not hinder the enzyme activity as a result of steric effects. The probes were able to detect amounts of phospholipase A2 as low as 0.0075 U mL(-1) and to monitor enzyme activity in real time. The probes were also used to screen phospholipase A2 inhibitors. For example, we found that the inhibition efficiency of MJ33 (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol) was higher than that of OBAA (3-(4-octadecyl)benzoylacrylic acid).
[Show abstract][Hide abstract] ABSTRACT: The photochemical behavior of Deepwater Horizon oil collected from the surface of the Gulf of Mexico was studied. Thin oil films on water were subjected to simulated sunlight, and the resulting chemical and optical changes were observed. Polycyclic aromatic hydrocarbons (PAHs) showed substantial photodegradation, with larger PAHs being more rapidly decomposed. About 60% of the fluorescence at the excitation and emission maxima was observed with 12h of simulated solar irradiation equivalent to approximately 3d of sunlight. Synchronous scan fluorescence measurements showed 80-90% loss of larger PAHs with 12h of simulated solar irradiation. Absorbance of the oil decreased by only 20% over the same time period. Alkanes showed no significant photochemical losses. After irradiation, the toxicity of water in contact with the oil significantly increased, presumably due to the release of water soluble photoproducts that were toxic. Photocatalyst addition resulted in enhanced degradation rate for PAHs, and toxicity of the aqueous layer was altered in the presence of photocatalysts added to the oil film. Photochemistry is an important pathway for degradation of large PAHs, which are typically resistant to biodegradation.
[Show abstract][Hide abstract] ABSTRACT: The surface of superparamagnetic silica coated iron oxide (Fe3O4@SiO2) nanoparticles was functionalized with a disulfide bond linked N-hydroxysuccinimidyl (NHS) ester group in order to develop a method for labeling primary amines in peptides/proteins. The nanoparticle labeled proteins/peptides formed after NHS ester reaction with the primary amine groups were isolated using a magnet without any additional purification step. Nanoparticle moieties conjugated to peptides/proteins were then trimmed by cleavage at the disulfide linker with a reducing agent. The labeled peptides were analyzed by LC-MS/MS to determine their sequences and the sites of NHS ester labeling. This novel approach allowed characterization of lysine residues on the solvent accessible surface of native bovine serum albumin. Low cost, rapid magnetic separation, and specificity towards primary amine groups make NHS ester coated Fe3O4@SiO2 nanoparticles a potential labeling probe to study proteins on living cell surfaces.
[Show abstract][Hide abstract] ABSTRACT: Photochemical production of hydroxyl radical from thin oil films over seawater and pure water was measured with benzoic acid as a selective chemical probe. Oil was collected from the surface of the Gulf of Mexico following the Deepwater Horizon spill. Benzoic acid reacts with hydroxyl radical to form hydroxybenzoic acids, with p-hydroxybenzoic acid (p-HBA) being the most abundant. Total hydroxyl radical formation was studied using high benzoic acid concentrations and varying exposure time. The total moles of hydroxyl radical produced in 24 hour irradiations of thin oil films over Gulf of Mexico water and pure water were 3.7 × 10-7 and 4.2 × 10-7 respectively. After initial tests were performed, titanium dioxide (TiO2) nanomaterials were added to the system in two different concentrations to study the effects of hydroxyl radical formation in the presence of a photocatalyst. At low concentrations, TiO2 nanoparticles showed an overall decrease in the total number of moles of hydroxyl radical formed in the oil systems with Gulf of Mexico water (2.5 × 10-7). Steady state concentration of photoproduction of hydroxyl radicals was determined. The concentration of benzoic acid was varied and exposure time was kept constant. In the no oxide oil systems, the steady state concentration was ten times the concentration found in previous literature for photoproduction of hydroxyl radical in unaltered sea water. Once a photocatalyst was added, the steady state concentration decreased predominantly due to an increase in the scavenging rate. This results means that there were other factors involved in the fate of the hydroxyl radical, and as a result of these factors, the hydroxyl radical did not penetrate effectively into the aqueous layer to react with benzoic acid. The oil in contact with the oxide surface played an important role in scavenging radicals before they could transfer into the aqueous phase. In addition, high concentrations of hydroxyl radical formed at the oxide surface may have enhanced self-reaction to form hydrogen peroxide, resulting in another scavenging pathway. This study provided important information into the formation and fate of the hydroxyl radical in thin oil films on water.
Semiconductor Photocatalysis, Solar Energy. Conversion and Advanced Oxidation Technologies (SP-SEC-AOTs-1), Cartagena, Colombia; 05/2013
[Show abstract][Hide abstract] ABSTRACT: Quantum dots (QDs) and magnetic nanoparticles (MNPs; typically 1–100 nm in dimension) have many applications in analytical methods. Quantum dots are semiconductor nanoparticles whose electronic energy levels are substantially controlled by the particle dimensions. This control comes about due to quantum confinement; that is, the size of the particle becomes small compared with the typical exciton size in the bulk material. An exciton is a hole-electron pair that has been spatially separated due to energy input. QDs have unique optical properties that make them useful as an analytical tool. These properties include broad absorbance spectra, narrow emission spectra, emission wavelength that is tunable by adjusting particle size, high quantum efficiency, and low photobleaching rates. Selectively attaching QDs to target analytes can effectively label the analyte for highly sensitive detection using optical or electrochemical methods. MNPs are commonly made of magnetite (Fe3O4) or maghemite (γ-Fe2O3). In the nanoscale range, these materials are typically superparamagnetic. The magnetic properties of these nanomaterials allow them to be manipulated by magnetic fields or detected by magnetic means. Furthermore, the relatively low toxicity of iron oxides allow for their use for in vivo applications. This review covers analytical applications of MNPs and QDs. The literature covered is mainly articles that appeared a few years before 2010 to emphasize advances contemporary to this review.
Encyclopedia of Analytical Chemistry, 12/2012; , ISBN: 0471976709
[Show abstract][Hide abstract] ABSTRACT: Vertically aligned Au–TiO2 core–shell nanowires were synthesized by using a two step method. Au nanowires were first synthesized using a galvanostatic constant current electrodeposition technique. A shell of anatase TiO2 was subsequently grown on the Au nanowires using pulsed laser deposition. The core–shell nanostructures were then characterized using electron microscopy, electron diffraction, and X-ray diffraction techniques. The results showed that the wires were highly aligned and well separated. Dye sensitized solar cells were then fabricated using the core–shell nanowire arrays as photoanode, N535 dye as the sensitizer and I3−/I− as the redox electrolyte. The Au nanowires inside the highly crystalline TiO2 anatase nanoshell provided a direct conduction path and improved transport for electrons between the TiO2 and the conducting substrate. This efficient electron conduction out of the oxide semiconductor enhanced the current generation as well as the power conversion efficiency of the cell. The influence of the TiCl4 post-treatment on Au–TiO2 core–shell nanowire electrodes is investigated and compared to nontreated films. Cell efficiencies are improved, due to higher photocurrents as a result of this post-treatment. Optical effects of the metal nanowire may have also contributed to improved performance.