Herein we firstly reported a simple, environment-friendly, controllable synthetic method of CuSe nanosnakes at room temperature using copper salts and sodium selenosulfate as the reactants, and bovine serum albumin (BSA) as foaming agent. As the amounts of selenide ions (Se2−) released from Na2SeSO3 in the solution increased, the cubic and snake-like CuSe nanostructures were formed gradually, the cubic nanostructures were captured by the CuSe nanosnakes, the CuSe nanosnakes grew wider and longer as the reaction time increased. Finally, the cubic CuSe nanostructures were completely replaced by BSA–CuSe nanosnakes. The prepared BSA–CuSe nanosnakes exhibited enhanced biocompatibility than the CuSe nanocrystals, which highly suggest that as-prepared BSA–CuSe nanosnakes have great potentials in applications such as biomedical engineering.
Copper selenide; Nanosnakes; Bovine serum albumin; Synthesis; Characterization; Mechanism; Biocompatibility
Klockmannite copper selenide nanosheets (CuSe NSs) are synthesized by a facile microwave-assisted method and fully characterized. The nanosheets have smooth surface and hexagonal shape. The lateral size is 200–500 nm × 400–800 nm and the thickness is 55 ± 20 nm. The current-voltage characteristics of CuSe NS films show unique Ohmic and high-conducting behaviors, comparable to the thermally-deposited gold electrode. The high electrical conductivity of CuSe NSs implies their promising applications in printed electronics and nanodevices. Moreover, the local electrical variation is observed, for the first time, within an individual CuSe NS at low bias voltages (0.1 ~ 3 V) by conductive atomic force microscopy (C-AFM). This is ascribed to the quantum size effect of NS and the presence of Schottky barrier. In addition, the influence of the molar ratio of Cu2+/SeO2, reaction temperature, and reaction time on the growth of CuSe NSs is explored. The template effect of oleylamine and the intrinsic crystal nature of CuSe NS are proposed to account for the growth of hexagonal CuSe NSs.
Comb-push Ultrasound Shear Elastography (CUSE) has recently been shown to be a fast and accurate two-dimensional (2D) elasticity imaging technique that can provide a full field-of- view (FOV) shear wave speed map with only one rapid data acquisition. The initial version of CUSE was termed U-CUSE because unfocused ultrasound push beams were used. In this paper, we present two new versions of CUSE – Focused CUSE (F-CUSE) and Marching CUSE (M-CUSE), which use focused ultrasound push beams to improve acoustic radiation force penetration and produce stronger shear waves in deep tissues (e.g. kidney and liver). F-CUSE divides transducer elements into several subgroups which transmit multiple focused ultrasound beams simultaneously. M-CUSE uses more elements for each focused push beam and laterally marches the push beams. Both F-CUSE and M-CUSE can generate comb-shaped shear wave fields that have shear wave motion at each imaging pixel location so that a full FOV 2D shear wave speed map can be reconstructed with only one data acquisition. Homogeneous phantom experiments showed that U-CUSE, F-CUSE and M-CUSE can all produce smooth shear wave speed maps with accurate shear wave speed estimates. An inclusion phantom experiment showed that all CUSE methods could provide good contrast between the inclusion and background with sharp boundaries while F-CUSE and M-CUSE require shorter push durations to achieve shear wave speed maps with comparable SNR to U-CUSE. A more challenging inclusion phantom experiment with a very stiff and deep inclusion shows that better shear wave penetration could be gained by using F-CUSE and M-CUSE. Finally, a shallow inclusion experiment showed that good preservations of inclusion shapes could be achieved by both U-CUSE and F-CUSE in the near field. Safety measurements showed that all safety parameters are below FDA regulatory limits for all CUSE methods. These promising results suggest that, using various push beams, CUSE is capable of reconstructing a 2D full FOV shear elasticity map using only one push-detection data acquisition in a wide range of depths for soft tissue elasticity imaging.
CUSE; comb-push; ultrasound elastography; shear wave; acoustic radiation force; unfocused ultrasound beam; focused ultrasound beam
In this study, the Mo-electrode thin films were deposited by a two-stepped process, and the high-purity copper indium selenide-based powder (CuInSe2, CIS) was fabricated by hydrothermal process by Nanowin Technology Co. Ltd. From the X-ray pattern of the CIS precursor, the mainly crystalline phase was CIS, and the almost undetectable CuSe phase was observed. Because the CIS powder was aggregated into micro-scale particles and the average particle sizes were approximately 3 to 8 μm, the CIS power was ground into nano-scale particles, then the 6 wt.% CIS particles were dispersed into isopropyl alcohol to get the solution for spray coating method. Then, 0.1 ml CIS solution was sprayed on the 20 mm × 10 mm Mo/glass substrates, and the heat treatment for the nano-scale CIS solution under various parameters was carried out in a selenization furnace. The annealing temperature was set at 550°C, and the annealing time was changed from 5 to 30 min, without extra Se content was added in the furnace. The influences of annealing time on the densification, crystallization, resistivity (ρ), hall mobility (μ), and carrier concentration of the CIS absorber layers were well investigated in this study.
Nano-scale particle; Spray coating method; CIS absorber layer; Annealing
The stability of protein drugs remains one of the key hurdles to their success in the market. The aim of the present study was to design a novel nanoemulsion drug-delivery system (NEDDS) that would encapsulate a standard-model protein drug – bovine serum albumin (BSA) – to improve drug stability.
The BSA NEDDS was prepared using a phase-inversion method and pseudoternary phase diagrams. The following characteristics were studied: morphology, size, zeta potential, drug loading, and encapsulation efficiency. We also investigated the stability of the BSA NEDDS, bioactivity of BSA encapsulated within the NEDDS, the integrity of the primary, secondary, and tertiary structures, and specificity.
The BSA NEDDS consisted of Cremophor EL-35, propylene glycol, isopropyl myristate, and normal saline. The average particle diameter of the BSA NEDDS was about 21.8 nm, and the system showed a high encapsulation efficiency (>90%) and an adequate drug-loading capacity (45 mg/mL). The thermodynamic stability of the system was investigated at different temperatures and pH levels and in room-temperature conditions for 180 days. BSA NEDDS showed good structural integrity and specificity for the primary, secondary, and tertiary structures, and good bioactivity of the loaded BSA.
BSA NEDDS showed the properties of a good nanoemulsion-delivery system. NEDDS can greatly enhance the stability of the protein drug BSA while maintaining high levels of drug bioactivity, good specificity, and integrity of the primary, secondary, and tertiary protein structures. These findings indicate that the nanoemulsion is a potential formulation for oral administration of protein drugs.
nanoemulsion; drug-delivery system; protein drug; oral administration; stability
AIM: To investigate the uptake difference between bovine serum albumin nanoparticle (BSA-NP) and bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (BSA-NP-GL) and to develop a novel hepatocyte targeting BSA-NP-GL based on active targeting technology mediated by specific binding site of GL on rat cellular membrane.
METHODS: Calcein loaded bovine serum albumin nanoparticles (Cal-BSA-NP) were prepared by desolvation process. Glycyrrhizin was conjugated to the surface reactive amino groups (SRAG) of Cal-BSA-NP by sodium periodate oxidization, which resulted in calcein-loaded bovine serum albumin nanoparticles with their surface modified by glycyrrhizin (Cal-BSA-NP-GL). The morphology of the two types of prepared nanoparticles (NP) was observed by transmission electron microscopy. The diameter of NP was measured with a laser particle size analyzer. The interaction between Cal-BSA-NP-GL and primary cultured hepatocytes was studied through cellular uptake experiments. The uptake amount of Cal-BSA-NP-GL and Cal-BSA-NP by rat hepatocytes was determined by fluorospectrophotometry. Uptake characteristics were investigated through experiments of competitive inhibition of specific binding site of GL.
RESULTS: Both Cal-BSA-NP-GL and Cal-BSA-NP had regular spherical surfaces. The average diameter of Cal-BSA-NP-GL and Cal-BSA-NP was 77 and 79 nm respectively. The uptake amount of the two NP by hepatocytes reached its maximum at 2 h after incubation. The uptake amount of Cal-BSA-NP-GL by rat hepatocytes was 4.43-fold higher than that of Cal-BSA-NP. There was a significant difference in the uptake of Cal-BSA-NP-GL and Cal-BSA-NP by hepatocytes (P<0.01). The uptake of Cal-BSA-NP-GL was inhibited when GL was added previously to isolated rat hepatocytes, and the uptake of Cal-BSA-NP was not affected by GL.
CONCLUSION: A binding site of GL is present on the surface of rat hepatocytes, BSA-NP-GL may be internalized via this site by hepatocytes and can be used as a drug carrier for active targeting of delivery drugs to hepatocytes.
Glycyrrhizin; Surface modified; Bovine serum albumin; Nanoparticles; Hepatocytes
Dye conjugation is a common strategy improving the surface enhanced Raman detection sensitivity of biomolecules. Reported is a proof-of-concept study of a novel surface enhanced Raman spectroscopic tagging strategy termed as acid-cleavable SERS tag (ACST) method. Using Rhodamine B as the starting material, we prepared the first ACST prototype that consisted of, from the distal end, a SERS tag moiety (STM), an acid-cleavable linker, and a protein reactive moiety. Complete acid cleavage of the ACST tags was achieved at a very mild condition that is 1.5% trifluoroacetic acid (TFA) aqueous solution at room temperature. SERS detection of this ACST tagged protein was demonstrated using bovine serum albumin (BSA) as the model protein. While the SERS spectrum of intact ACST-BSA was entirely dominated by the fluorescent signal of STM, quality SERS spectra can be readily obtained with the acid cleaved ACST-BSA conjugates. Separation of the acid cleaved STM from protein further enhances the SERS sensitivity. Current SERS detection sensitivity, achieved with the acid cleaved ACST-BSA conjugate is ~5 nM in terms of the BSA concentration and ~1.5 nM in ACST content. The linear dynamic range of the cleaved ACST-BSA conjugate spans four orders of magnitudes from ~10 nM to ~100 μM in protein concentrations. Further improvement in the SERS sensitivity can be achieved with resonance Raman acquisition. This cleavable tagging strategy may also be used for elimination of protein interference in fluorescence based biomolecule detection.
The livertaxis of glycyrrhizic acid-conjugated bovine serum albumin (GL-BSA) has been reported in the literature. Now, in this paper, we describe a novel type of drug-targeted delivery system containing 10-hydroxycamptothecin (HCPT) with liver tumor targeting.
First, GL was coupled to BSA then HCPT was encapsulated in GL-BSA by high-pressure homogenization emulsification. In the experimental design, the influencing variables on particle size and drug loading efficiency were determined to be BSA concentration, volume ratio of water to organic phase, and speed and speed duration of homogenization as well as homogenization pressure and the number of times homogenized at certain pressures. Particle size plays an important role in screening optimal conditions of nanoparticles preparation. Characteristics of 10-hydroxycamptothecin-loaded glycyrrhizic acid-conjugated bovine serum albumin nanoparticles (GL-BSA-HCPT-NPs), such as the drug encapsulation efficiency, drug loading efficiency, and GL-BSA content were studied. In addition, the morphology of the nanoparticles (NPs) and weight loss rate were determined and Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, and thermal analysis performed.
The average particle size of the sample NPs prepared under optimal conditions was 157.5 nm and the zeta potential was −22.51 ± 0.78 mV; the drug encapsulation efficiency and drug loading efficiency were 93.7% and 10.9%, respectively. The amount of GL coupling to BSA was 98.26 μg/mg. Through physical property study of the samples, we determined that the HCPT had been successfully wrapped in GL-BSA. In vitro drug-release study showed that the nanoparticles could release the drug slowly and continuously. Hemolysis testing showed the safety of GL-BSA as a novel drug delivery system. The targeting properties of GL-BSA-HCPT-NPs were studied in an in vitro cell uptake study and cell proliferation assay. Cells incubated with GL-BSA-HCPT-NPs and labeled with fluorescein isothiocyanate showed more extensive fluorescence spots and stronger fluorescence intensity than samples without GL conjugation. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine the inhibitory rate of the samples. It was found that the inhibitory rate of GL-BSA-HCPT-NPs develops as concentration rises. Further, the inhibitory rate of GL-BSA-HCPT-NPs was higher at the same concentration and had a lower half maximal inhibitory concentration value than the other samples. The half maximal inhibitory concentration values of GL-BSA-HCPT-NPs, BSA-HCPT-NPs, and HCPT sodium were 0.78 ± 0.015, 1.62 ± 0.039, and 7.93 ± 0.255 μg/mL, respectively.
The results of this study show GL-BSA-HCPT to be a promising new vehicle for hepatocellular carcinoma-targeting therapy.
HCPT; BSA; GL; high-pressure homogenization emulsification
This document reports a novel method of measuring association rate constant (ka) for antibody-antigen interaction using evanescent wave-based combination tapered fiber-optic biosensor (CTFOB) dip-probes. The method was demonstrated by measuring association rate constant for bovine serum albumin (BSA) and anti-BSA antibody interaction. “Direct method” was used for detection; goat anti-BSA “capture” antibodies were immobilized on the probe surfaces while the antigen (BSA) was directly labeled with Alexa 488 dye. The probes were subsequently submerged in 3 nM Labeled BSA in egg albumin (1 mg/ml). The fluorescence signal recorded was proportional to BSA anti-BSA conjugates and continuous signal was acquired suing a fiber optic spectrometer (Ocean Optics, Inc.). A 476 nm diode laser was use as an excitation source. Association constant was estimated from a plot of signal as a function of time. Measured association rate constant ka for the binding of BSA with anti-BSA at room temperature is (8.33 ± 0.01) ×104 M−1 s−1.
association constant; BSA; anti-BSA; fiber-optic; biosensor; real-time
Background and the purpose of the study
The binding ability of a drug to serum albumin has influence on the pharmacokinetics of a drug. In the present study, the mutual interaction of anticancer drug erlotinib hydrochloride with bovine serum albumin (BSA) using fluorescence and UV/vis spectroscopy was investigated.
The BSA solution (0.1 mM) was prepared daily in Tris buffer (0.05 mol l-1, pH =7.4) and treated at final concentration of 1.67×10-5 M with different amounts of erlotinib hydrochloride to obtain final concentrations of 0, 0.2, 0.4, 0.8, 1, 2, 4, 6, 8, 20 and 42 µM receptively. The mixture was allowed to stand for 5 min and the fluorescence quenching spectra were recorded at 298, 303, 308 and 313 K.
It was found that erlotinib hydrochloride caused the fluorescence quenching of BSA by the formation of a BSA-erlotinib hydrochloride complex. The mechanism of the complex formation was then analyzed by determination of the number of binding sites, apparent binding constant K, and calculation of the corresponding thermodynamic parameters such as the free energy (△G), enthalpy (△H) and entropy changes (△S) at different temperatures. Results showed that binding of erlotinib hydrochloride to BSA was spontaneous, and the hydrophobic forces played a major role in the complex formation. The distance, r, between donor (BSA) and acceptor (erlotinib hydrochloride) was found to be less than 8 nm suggesting the occurrence of non-radiative energy transferring and static quenching between these two molecules.
The results provided preliminary information on the binding of erlotinib hydrochloride to BSA and the presence of a single binding site on BSA and K values for the association of BSA with erlotinib hydrochloride increased by the increase in temperature.
Erlotinib hydrochloride; Albumin; Fluorescence quenching
Bovine serum albumin (BSA) contains high affinity binding sites for several endogenous and exogenous compounds and has been used to replace human serum albumin (HSA), as these two compounds share a similar structure. Naringin palmitate is a modified product of naringin that is produced by an acylation reaction with palmitic acid, which is considered to be an effective substance for enhancing naringin lipophilicity. In this study, the interaction of naringin palmitate with BSA was characterised by spectroscopic and molecular docking techniques.
The goal of this study was to investigate the interactions between naringin palmitate and BSA under physiological conditions, and differences in naringin and naringin palmitate affinities for BSA were further compared and analysed. The formation of naringin palmitate-BSA was revealed by fluorescence quenching, and the Stern-Volmer quenching constant (KSV) was found to decrease with increasing temperature, suggesting that a static quenching mechanism was involved. The changes in enthalpy (ΔH) and entropy (ΔS) for the interaction were detected at −4.11±0.18 kJ·mol−1 and −76.59±0.32 J·mol−1·K−1, respectively, which indicated that the naringin palmitate-BSA interaction occurred mainly through van der Waals forces and hydrogen bond formation. The negative free energy change (ΔG) values of naringin palmitate at different temperatures suggested a spontaneous interaction. Circular dichroism studies revealed that the α-helical content of BSA decreased after interacting with naringin palmitate. Displacement studies suggested that naringin palmitate was partially bound to site I (subdomain IIA) of the BSA, which was also substantiated by the molecular docking studies.
In conclusion, naringin palmitate was transported by BSA and was easily removed afterwards. As a consequence, an extension of naringin applications for use in food, cosmetic and medicinal preparations may be clinically and practically significant, especially in the design of new naringin palmitate-inspired drugs.
Hollow hydroxyapatite (HA) microspheres were prepared by reacting solid microspheres of Li2O–CaO–B2O3 glass (106–150 μm) in K2HPO4 solution, and evaluated as a controlled delivery device for a model protein, bovine serum albumin (BSA). Reaction of the glass microspheres for 2 days in 0.02 M K2HPO4 solution (pH = 9) at 37°C resulted in the formation of biocompatible HA microspheres with a hollow core diameter equal to 0.6 the external diameter, high surface area (~100 m2/g), and a mesoporous shell wall (pore size ≈13 nm). After loading with a solution of BSA in phosphate-buffered saline (PBS) (5 mg BSA/ml), the release kinetics of BSA from the HA microspheres into a PBS medium were measured using a micro bicinchoninic acid (BCA) protein assay. Release of BSA initially increased linearly with time, but almost ceased after 24–48 h. Modification of the BSA release kinetics was achieved by modifying the microstructure of the as-prepared HA microspheres using a controlled heat treatment (1–24 h at 600–900°C). Sustained release of BSA was achieved over 7–14 days from HA microspheres heated for 5 h at 600°C. The amount of BSA released at a given time was dependent on the concentration of BSA initially loaded into the HA microspheres. These hollow HA microspheres could provide a novel inorganic device for controlled local delivery of proteins and drugs.
We report a new approach to colorimetric Enzyme-Linked Immunosorbent Assay (ELISA) that reduces the total assay time to < 2 min and the lower-detection-limit by 100-fold based on absorbance readout. The new approach combines the use of silver nanoparticles, microwaves and split ring resonators (SRR). The SRR structure is comprised of a square frame of copper thin film (30 µm thick, 1 mm wide, overall length of ~9.4 mm on each side) with a single split on one side, which was deposited onto a circuit board (2×2 cm2). A single micro-cuvette (10 µl volume capacity) was placed in the split of the SRR structures. Theoretical simulations predict that electric fields are focused in and above the micro-cuvette without the accumulation of electrical charge that breaks down the copper film. Subsequently, the walls and the bottom of the micro-cuvette were coated with silver nanoparticles using a modified Tollen’s reaction scheme. The silver nanoparticles served as a mediator for the creation of thermal gradient between the bioassay medium and the silver surface, where the bioassay is constructed. Upon exposure to low power microwave heating, the bioassay medium in the micro-cuvette was rapidly and uniformly heated by the focused electric fields. In addition, the creation of thermal gradient resulted in the rapid assembly of the proteins on the surface of silver nanoparticles without denaturing the proteins. The proof-of-principle of the new approach to ELISA was demonstrated for the detection of a model protein (biotinylated-bovine serum albumin, b-BSA). In this regard, the detection of b-BSA with bulk concentrations (1 µM to 1 pM) was carried out on commercially available 96-well high throughput screening (HTS) plates and silver nanoparticle-deposited SRR structures at room temperature and with microwave heating, respectively. While the room temperature bioassay (without microwave heating) took 70 min to complete, the identical bioassay took < 2 min to complete using the SRR structures (with microwave heating). A lower detection limit of 0.01 nM for b-BSA (100-fold lower than room temperature ELISA) was observed using the SRR structures.
Immunoassays; Bioassays; ELISA; Silver island films; Silver colloids
The effects of nonglycated bovine serum albumin (BSA) and advanced glycosylation end products of BSA (AGE-BSA) on vascular responses of control and metabolic syndrome (MS) rats characterized by hypertriglyceridemia, hypertension, hyperinsulinemia, and insulin resistance were studied. Albumin and in vitro prepared AGE-BSA have vascular effects; however, recent studies indicate that some effects of in vitro prepared AGEs are due to the conditions in which they were generated. We produced AGEs by incubating glucose with BSA for 60 days under sterile conditions in darkness and at 37°C. To develop MS rats, male Wistar animals were given 30% sucrose in drinking water since weanling. Six month old animals were used. Blood pressure, insulin, triglycerides, and serum albumin were increased in MS rats. Contraction of aortic rings elicited with norepinephrine was stronger. There were no effects of nonglycated BSA or AGE-BSA on contractions in control or MS rats; however, both groups responded to L-NAME, an inhibitor of nitric oxide synthesis. Arterial relaxation induced using acetylcholine was smaller in MS rats. Nonglycated BSA and AGE-BSA significantly diminished relaxation in a 35% in the control group but the decrease was similar when using nonglycated BSA and AGE-BSA. This decrease was not present in the MS rats and was not due to increased RAGEs or altered biochemical characteristics of BSA. In conclusion, both BSA and AGE-BSA inhibit vascular relaxation in control artic rings. In MS rats the effect is lost possibly due to alterations in endothelial cells that are a consequence of the illness.
AGE-BSA; BSA; metabolic syndrome; vascular relaxation
Thymoquinone (TQ), an active component of Nigella sativa L., is known to have anti-cancer and anti-inflammatory effects; however, no studies on its analytical detection in serum and its protein binding have been published. Using high performance liquid chromatography analysis, we show that the average recovery of TQ from serum is 2.5% at 10 μg/ml of TQ and 72% at 100 μg/ml. The low recovery of TQ from serum is due to its extensive binding to plasma proteins, as more than 99% of TQ was bound within 30 min of incubation. The binding of TQ to the major plasma proteins, bovine serum albumin (BSA) and alpha −1 acid glycoprotein (AGP), was studied and found to be 94.5 ± 1.7% for BSA and 99.1 ± 0.1% for AGP. Mass spectrometric analysis revealed that TQ was bound covalently to BSA, specifically on Cyst-34. Using WST-1 proliferation assay, we showed that BSA plays a protective role against TQ-induced cell death; pre-incubation with BSA prevented TQ from exerting its anti-proliferative effects against DLD-1 and HCT-116 human colon cancer cells. On the other hand, binding of TQ to AGP did not alter its anti-proliferative activity against both cell lines. When TQ was pre-incubated with AGP prior to the addition of BSA, the activity of TQ against DLD-1 was maintained, suggesting that AGP prevented the binding of TQ to BSA. This is the first time the covalent binding and inhibitory effect of BSA on TQ is documented. These data offer new grounds for TQ future pharmacokinetic analysis in vivo.
Electronic supplementary material
The online version of this article (doi:10.1007/s12154-010-0052-4) contains supplementary material, which is available to authorized users.
Thymoquinone; Mass spectrometry; Serum; Protein binding; Anticancer activity
To test the moderating effects of decision-making style and gender on the relationship between condom use self-efficacy (CUSE) and condom use behavior among sexually active adolescents and young adults.
Twenty-four continuation high schools in California.
Data were collected between February 2008 to June 2009 from a sample of 1304 sexually active adolescents and young adults. The mean (SD) age of the participants was 16.8 (0.9) years, 41% were female, and the ethnicity frequencies were Hispanic, 65%; mixed, 13%; white, 11%; black, 6%; and other, 5%.
The tools used were CUSE, decision-making–self-confidence, and decision-making–approach.
Main Outcome Measure
Condom use during the most recent sexual intercourse (termed last sex).
Forty-five percent of sexually active participants used condoms at last sex. Decision-making–self-confidence and decision-making–approach significantly moderated the effect of CUSE on condom use. The positive relationship between CUSE and condom use was relatively stronger for males and females reporting high vs low decision-making–self-confidence. Among females, the relationship between CUSE and condom use at last sex was weaker for those reporting high vs low decision-making–approach. Both of these effects were observed at high levels of CUSE.
Programs for sexually transmitted infection/human immunodeficiency virus prevention including CUSE content may increase adolescent and young adult condom use by targeting interventions to decision-making style and gender.
Neoglycoconjugate vaccines synthesized by the squaric acid spacer method allow single point attachment of the carbohydrate antigen to the protein carrier. However, the localization of the carbohydrate antigen sites of conjugation on the protein carrier has been an elusive task difficult to achieve.
Covalent attachment of the lactose antigen to the bovine serum albumin (BSA) was prepared by the squaric acid method using a hapten:BSA ratio of 20:1. Different reaction times were used during the conjugation reaction and two different lactose-BSA glycoconjugate vaccines were obtained. The carbohydrate antigen hapten:BSA ratios of these lactose-BSA glycoconjugate vaccines were determined by MALDI-TOF/RTOF-MS and the glycation sites in the neoglycoconjugates were determined using nano-LC/ESI-QqTOF-MS/MS analysis of the trypsin and GluC V8 digests of the conjugates.
We have identified a total of 15 glycation sites located on the BSA lysine residues for the neoglycoconjugate vaccine formed with a hapten:BSA ratio of 5.1:1, However, the tryptic and GluC V8 digests of the hapten-BSA glycoconjugate with a hapten:BSA ratio of 19.0:1 allowed identification of 30 glycation sites located on the BSA. These last results seem to indicate that this conjugation results in formation of various glycoforms.
It was observed that the number of identified glycation sites increased when the hapten:BSA ratio of glycoconjugate formation increased, and that the location of the glycation sites appears to be mainly on the outer surface of the BSA carrier molecule which is in line with the assumption that the sterically more accessible lysine residues, namely those located on the outer surface of the BSA, would be conjugated preferentially.
Bovine serum albumin (BSA) protected nanoclusters (Au and Ag) represent a group of nanomaterials that holds great promise in biophysical applications due to their unique fluorescence properties and lack of toxicity. While, these metal nanoclusters have -utility in a variety of disciplines including catalysis, biosensing, photonics, imaging and molecular electronics. However, they suffer from several certain disadvantages such as low fluorescence quantum efficiency (typically near 6%) and broad emission spectrum (540nm to 800nm). We describe an approach to enhance the apparent brightness of BSA Au clusters by linking it with high extinction donor organic dye pacific blue (PB). In this conjugate PB acts as a donor to BSA Au clusters and enhances its brightness by resonance energy transfer (RET). We found that the emission of BSA Au clusters can be enhanced by a magnitude of two-folds by resonance energy transfer (RET) from the high extinction donor PB, and BSA Au clusters can act as an acceptor to nanosecond lifetime organic dyes. By pumping the BSA Au clusters using a high extinction donor, one can increase the effective brightness of less bright fluorophores like BSA Au clusters. Moreover, we prepared another conjugate of BSA Au clusters with the near infra-red (NIR) dye Dylight 750 (Dy750), where BSA Au cluster act as a donor to Dy750. We observed that BSA Au clusters can function as a donor, showing 46% transfer efficiency to the NIR dye Dy750 with long lifetime component in acceptor decay through RET. Such RET-based probes can be used to prevent the problems of broad emission spectrum associated with the BSA Au clusters. Moreover, transferring energy from BSA Au cluster to Dy750 will result in a RET probe with narrow emission spectrum and long lifetime component which can be utilized in imaging applications.
A potential new treatment for drug addiction is immunization with vaccines that induce antibodies that can abrogate the addictive effects of the drug of abuse. One of the challenges in the development of a vaccine against drugs of abuse is the availability of an optimum procedure that gives reproducible and high yielding hapten-protein conjugates. In this study, a heroin/morphine surrogate hapten (MorHap) was coupled to bovine serum albumin (BSA) using maleimide-thiol chemistry. MorHap-BSA conjugates with 3, 5, 10, 15, 22, 28, and 34 haptens were obtained using different linker and hapten ratios. Using this optimized procedure, MorHap-BSA conjugates were synthesized with highly reproducible results and in high yields. The number of haptens attached to BSA was compared by 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, modified Ellman’s test and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Among the three methods, MALDI-TOF MS discriminated subtle differences in hapten density. The effect of hapten density on enzyme-linked immunosorbent assay (ELISA) performance was evaluated with seven MorHap-BSA conjugates of varying hapten densities, which were used as coating antigens. The highest antibody binding was obtained with MorHap-BSA conjugates containing 3–5 haptens. This is the first report that rigorously analyzes, optimizes and characterizes the conjugation of haptens to proteins that can be used for vaccines against drugs of abuse. The effect of hapten density on the ELISA detection of antibodies against haptens demonstrates the importance of careful characterization of the hapten density by the analytical techniques described.
Electronic supplementary material
The online version of this article (doi:10.1007/s00216-014-8035-x) contains supplementary material, which is available to authorized users.
Hapten density; Drugs of abuse vaccines; TNBS; Ellman’s test; MALDI-TOF MS; ELISA
Background and Objectives
Laser-activated photodynamic biologic tissue glues may be useful for closing incisions in ophthalmology. We report on the use of two such preparations to close perforating corneal incisions in living rats.
Study Design/Materials and Methods
A previously described preparation containing a covalent albumin-chlorin e6 (ce6) conjugate (bovine serum albumin (BSA)–ce6), and a novel mixture of albumin and Janus Green (BSA/JG), both activated with a 665-nm diode laser were used to glue mouse skin ex vivo. The optimized glues were then used to seal incisions in rat corneas and results were compared to control incisions. Rats were sacrificed at day 1, 7, and 14 and eyes tested for leaking pressure and examined histopathologically.
One day after treatment eyes closed with BSA–ce6 had a leaking pressure (in mmHg) of 357 compared to 193 for control incisions (P<0.01); closure with BSA/JG gave a leaking pressure of 430 (P<0.05 compared to BSA–ce6, and P<0.001 compared to control). Histological examination showed eyes sealed with BSA/JG have less inflammation present than untreated eyes at 7 days.
These data demonstrate that photodynamic laser activated tissue glues can be used to effectively seal corneal incisions in living animals without thermal damage or undue inflammation.
Photodynamic therapy; tissue glue; laser activation; rat corneal incision; Janus Green
Nanocrystalline lead selenide (PbSe) thin films were prepared on glass substrates by a chemical bath deposition method, using sodium selenosulfate (Na2SeSO3) as a source of Se2− ions, and lead acetate as a source of Pb2+ ions. Trisodium citrate (TSC) was used as a complexing agent. PbSe films were prepared at various deposition temperatures while the pH value was kept fixed at 11, and the effect on the resulting film properties was studied by X-ray diffraction (XRD), X-ray fluorescence (XRF), scanning electron microscopy (SEM) and optical absorption studies. The structural parameters, such as the lattice constant (a), crystallite size (D), dislocation density (ρ) and microstrain (ε) were evaluated from the XRD spectra. It was found that average crystallite size, as calculated from Scherrer’s formula, increased from 23 to 33 nm as the deposition temperature was varied from 303 to 343 K. The dislocation density and microstrain were found to vary inversely with the crystallite size, whereas the lattice constant was found to increase with an increase in crystallite size. The optical absorption spectra of the nanocrystalline PbSe films showed a blue shift, and the optical band gap (E
) was found to increase from 1.96 to 2.10 eV with the decrease in crystallite size.
chemical bath deposition; lattice parameter; lead selenide; Nelson–Riley plot; optical absorption
Implants that simultaneously function as an osteoconductive matrix and as a device for local drug or growth factor delivery could provide an attractive system for bone regeneration. In our previous work, we prepared hollow hydroxyapatite (abbreviated HA) microspheres with a high surface area, mesoporous shell wall and studied the release of a model protein, bovine serum albumin (BSA), from the microspheres into phosphate-buffered saline (PBS). The present work is an extension of our previous work to study the release of BSA from similar HA microspheres into a biocompatible hydrogel, poly(ethylene glycol) (PEG). BSA-loaded HA microspheres were placed in a PEG solution which was rapidly gelled using ultraviolet radiation. The BSA release rate into the PEG hydrogel, measured using a spectrophotometric method, was slower than into PBS, and it was dependent on the initial BSA loading and on the microstructure of the microsphere shell wall. A total of 35–40% of the BSA initially loaded into the microspheres was released into PEG over ~14 days. The results indicate that these hollow HA microspheres have promising potential as an osteoconductive device for local drug or growth factor delivery in bone regeneration and in the treatment of bone diseases.
Hollow hydroxyapatite microspheres; controlled release; bovine serum albumin; poly(ethylene glycol) hydrogel
We examined the role of antigenic electrical charge as a determinant of glomerular immune complex localization in the rabbit. Serum sickness nephritis was induced in groups of New Zealand white rabbits by daily 25-mg intravenous injections of bovine serum albumin (BSA) chemically modified to be cationic (pI > 9.5) or more anionic (pI, 3.5-4.6); an additional group received unmodified native BSA (pI, 4.5-5.1). Factors known to influence immune complex localization, e.g., molecular size of the administered antigen and resulting circulating immune complexes, immunogenicity, and disappearance time from the circulation were examined and found to be similar for both anionic and cationic BSA. Charge modification did increase the nonimmune clearance of cationic and anionic BSA compared with native BSA. Injected cationic BSA was shown in paired label experiments to bind directly to glomeruli compared with native BSA. The renal lesion produced by cationic BSA was markedly different from that found in rabbits given anionic or native BSA. Animals receiving cationic BSA uniformly developed generalized diffuse granular capillary wall deposits of IgG, C3, and BSA detected after 2 wk of injections and increasing until death at 6 wk. Qualitatively similar deposits were produced by the administration of low doses of cationic BSA of only 1 or 10 mg/d. In contrast, the injection of both anionic and native BSA resulted in mesangial deposits at 2 and 4 wk with capillary wall deposits appearing by 6 wk. Ultrastructural examination of animals receiving cationic BSA revealed pure, extensive formation of dense deposits along the lamina rara externa of the glomerular basement membrane whereas such deposits were absent or rare in animals injected with the anionic or native BSA. Albuminuria was significantly greater at 6 wk in the groups receiving cationic BSA with a mean of 280 mg/24 h compared with 53 mg/24 h in the combined groups injected with anionic or native BSA. Blood urea nitrogen values were similar in all groups at 2 and 4 wk but higher in the animals receiving cationic BSA at 6 wk.
These experiments describe the reproducible induction of epimembranous immune deposits by administration of an exogenous cationic antigen. They suggest that antigenic charge can play an important role in the pathogenesis of membranous nephropathy by permitting direct glomerular binding of an antigen and predisposing to in situ immune complex formation.
In clinical practice, an overwhelming majority of biopsied thyroid nodules are benign. Therefore, there is a need for a complementary and noninvasive imaging tool to provide clinically relevant diagnostic information about thyroid nodules to reduce the rate of unnecessary biopsies. The goal of this study was to evaluate the feasibility of utilizing Comb-push Ultrasound Shear Elastography (CUSE) to measure the mechanical properties (i.e., stiffness) of thyroid nodules and use this information to help classify nodules as benign or malignant. CUSE is a fast and robust 2D shear elastography technique in which multiple laterally distributed acoustic radiation force beams are utilized simultaneously to produce shear waves. Unlike other shear elasticity imaging modalities, CUSE does not suffer from limited field of view (FOV) due to shear wave attenuation and can provide a large FOV at high frame rates. To evaluate the utility of CUSE in thyroid imaging, a preliminary study was performed on a group of 5 healthy volunteers and 10 patients with ultrasound (US)-detected thyroid nodules prior to fine needle aspiration biopsy (FNAB). The measured shear wave speeds in normal thyroid tissue and thyroid nodules were converted to Young's modulus (E), indicating a measure of tissue stiffness. Our results indicate an increase in E for thyroid nodules compared to normal thyroid tissue. This increase was significantly higher in malignant nodules compared to benign. The Young's modulus in normal thyroid tissue, benign and malignant nodules were found to be 23.2±8.29 kPa, 91.2±34.8 kPa, and 173.0±17.1 kPa, respectively. Results of this study suggest the utility of CUSE in differentiating between benign and malignant thyroid nodules.
Cancer; Elasticity; In vivo; Shear Wave Elastography; Thyroid; Ultrasound
The binding of proteins to a nanostructure often alters protein secondary and tertiary structures. However, the main physical mechanisms that elicit protein conformational changes in the presence of the nanostructure have not yet been fully established. Here we performed a comprehensive spectroscopic study to probe the interactions between bovine serum albumin (BSA) and carbon-based nanostructures of graphene and single-walled carbon nanotubes (SWNTs). Our results showed that the BSA “corona” acted as a weak acceptor to facilitate charge transfer from the carbon nanostructures. Notably, we observed that charge transfer occurred only in the case of SWNTs but not in graphene, resulting from the sharp and discrete electronic density of states of the former. Furthermore, the relaxation of external α–helices in BSA secondary structure increased concomitantly with the charge transfer. These results may help guide controlled nanostructure-biomolecular interactions and prove beneficial for developing novel drug delivery systems, biomedical devices and engineering of safe nanomaterials.
Protein corona; charge transfer; graphene; carbon nanotubes; Raman spectroscopy; FTIR spectroscopy