Bovine Serum Albumin Conjugate Research Articles

Modulated Protein Binding Ability of Anti-Diabetic Drugs in Presence of Monodispersed Gold Nanoparticles and its Inhibitory Potential towards Advanced Glycated End (AGE) Product Formation.

Binding strength of the anti-diabetic drugs chlorpropamide (CPM) and tolbutamide (TBM) with model protein bovine serum albumin (BSA) shows strong modulation in presence of colloidal gold nanoparticles (AuNP). Intrinsic tryptophan fluorescence of both the native BSA and BSA-AuNP conjugate quenched in presence of the drugs. Stern-Volmer quenching constant (KSV) of CPM binding to BSA-AuNP conjugate at different temperatures is almost twice (6.76~14.76 × 103M-1) than the corresponding values in native BSA (3.21~5.72 × 103M-1). However, the calculated KSV values with TBM show certain degree of reduction in presence of AuNP (6.46× 103M-1), while comparing with native BSA (8.83 × 103M-1). The binding mode of CPM towards BSA-AuNP conjugate is mainly through hydrophobic forces; whereas, TBM binding is identified to be Van der Waal's and hydrogen bonding type of interaction. Fluorescence lifetime analysis confirms static type of quenching for the intrinsic tryptophan fluorescence of BSA as well as BSA-AuNP conjugate with addition of CPM and TBM at different concentrations. The α-helical content in the secondary structure of BSA is decreased to 48.32% and 45. 28% in presence of AuNP, when the concentration of CPM is 0.08mM and 0.16mM in comparison with that of native protein (50.13%). On the other hand, the intensity of sugar induced advanced glycated end (AGE) product fluorescence is decreased by 55% and 80% at 0.13nM and 0.68nM AuNP, respectively. Change in the binding strength of the drugs with transport protein and reduced AGE product formation in presence of AuNP could lead to a major development in the field of nanomedicine and associated drug delivery techniques. Graphical Abstract Modulated drug binding ability and AGE product formation of serum proteins in presence of AuNP.

Bovine Serum Albumin Amplified Reactive Oxygen Species Generation from Anthrarufin-Derived Carbon Dot and Concomitant Nanoassembly for Combination Antibiotic–Photodynamic Therapy Application

Amplification of reactive oxygen species (ROS) generation through covalent conjugation of bovine serum albumin (BSA) with newly synthesized, ROS-producing carbon dots (CDs) upon visible light irradiation is reported for the first time. Derivatization of surface carboxyl functional groups of Anthrarufin-derived, green-emitting CD with the amine functionality of BSA ushers distinct changes in the photophysics of CD including an unprecedented ∼50 nm shift in its excitation maxima, decrease in fluorescence lifetime, and concomitant increase in ROS generation. Substantial conformational changes of BSA were witnessed upon conjugation with CD, rendering the BSA-CD conjugate resistant to pepsinolysis. A protease-proof nanoassembly was derived from the BSA-CD conjugate through desolvation that simultaneously hosts a prototype antibiotic and generates ROS with excellent efficiency, making it an attractive platform for antibacterial photodynamic therapy (A-PDT) applications. Systemic annihilation of both Gram-positive and -negative bacteria was achieved with the BSA-CD nanoassembly and envisioned as alternatives to traditional photosensitizers.

Supramolecular Assembly of a Molecularly Engineered Protein and Polymer.

Programmable assembly of biomolecules is a fast growing research area that aims to emulate nature's elegance in creating numerous hierarchical self-assembled structures, which are responsible for unimaginably difficult biological functions. Protein assembly is a particularly challenging task, owing to their structural diversity, conformational heterogeneity, and high molecular weight. This article reveals the ability of a supramolecular structure-directing unit (SSDU) to regulate the entropically favourable supramolecular assembly of a covalently conjugated protein (bovine serum albumin (BSA)) to produce well-defined protein-decorated micelles with remarkably high thermal stability, suppression of the thermal denaturation of the protein, and retention of enzymatic activity. Furthermore, a SSDU-appended thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) co-assembles with the SSDU-BSA conjugate because, in both cases, assembly was primarily driven by specific molecular recognition between the SSDUs. However, the resulting supramolecular protein-polymer conjugate exhibits distinctly different polymersome structure to that of the micellar particle produced by the protein-SSDU conjugate. In this case, the enzymatic activity can be significantly suppressed above the lower critical solution temperature of supramolecularly conjugated PNIPAM, possibly due to collapse of the de-solvated polymer chains on the protein surface.

Glucocorticoid receptor involvement in goose (Anas cygnoides) pituitary somatotroph differentiation induced by glucocorticoids during embryonic development

ABSTRACT 1. In this study, geese (Anas cygnoides) embryonic pituitary cells were cultured in vitro to determine if glucocorticoids could induce growth hormone (GH) expression and to investigate the molecular mechanisms involved in this process. 2. On embryonic day 15 (e15) and e20 the pituitary cells were treated with corticosterone (CORT), membrane impermeable bovine serum albumin–conjugate corticosterone (CORT-BSA), dexamethasone (DEX), and a glucocorticoid receptor (GR) antagonist (RU486) to detect responsiveness of somatotrophs to glucocorticoids. 3. Treatment with CORT, CORT-BSA, and DEX for as little as 6 h increased the percentage of GH-positive cells (P < 0.01) and increased GH mRNA expression (P < 0.01) in e15 goose pituitary cells compared to the control. CORT significantly increased the level of GH protein secreted from cultured e15 goose embryonic pituitary cells, and CORT-BSA increased GH secretion from e20 goose embryonic pituitary cells. 4. A significant increase was observed in the glucocorticoid receptor in GR transcription levels (P < 0.01) with CORT, CORT-BSA, and DEX treatment. Furthermore, the CORT-stimulated GH mRNA expression was completely negated by pre-treatment with RU486. 5. These findings demonstrate that glucocorticoids can stimulate somatotroph differentiation in vitro, as characterised by enhanced GH protein secretion andmRNA expression in cultured geese embryonic pituitary cells. The membrane GR was involved in pituitary somatotroph differentiation induced by glucocorticoids during the embryonic development of geese.

Development of Functional Antibodies Directed to Human Dialyzable Leukocyte Extract (Transferon®)

Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product.

Development of a progesterone-specific IgE assay for diagnosing patients with suspected progestogen hypersensitivity

BackgroundProgesterone hypersensitivity (PH) manifests as a spectrum of allergic symptoms during the luteal phase of the menstrual cycle. Confirming progesterone-specific immunoglobulin E (IgE; sIgE) by skin testing is unreliable because of irritant responses. ObjectiveTo develop a progesterone sIgE assay to assist in diagnosing PH. MethodsA progesterone–bovine serum albumin (BSA) conjugate was characterized and used to analyze sera collected from women in our center with suspected PH in a 1-batch enzyme-linked immunosorbent assay (ELISA) to establish high, low and negative cut-points. Sera collected from healthy nonatopic female subjects and from women with classical PH symptoms were included as negative and positive controls, respectively. Values exceeding the average negative control (OD + 3× the standard deviation) were considered positive. These cut-points were subsequently used to establish positive and negative results for serum from women with suspected PH received from other centers. A subset of high positive sera was used for ELISA-inhibition and in a beta-hexosaminidase mediator release assay to evaluate the specificity and functional relevance of progesterone-specific serum IgE, respectively. The numbers of true negative, false negative, true positive, and false positive samples were determined. ResultsThe direct progesterone sIgE ELISA results ranged from high positive to low positive and negative compared with healthy nonatopic control sera. Enzyme-linked immunosorbent assay inhibition and beta-hexosaminidase mediator release confirmed specificity and functional relevance of progesterone-sIgE, respectively. The sensitivity, specificity positive predictive values and negative predictive values were found to be 82%, 100%, 86%, and 100%, respectively, using the mediator release assay results as the gold standard. ConclusionThis assay has a good specificity and positive predictive value for screening women with suspected PH for progesterone sIgE.

Label-free gold nanoclusters as quenchable fluorescent probes for sensing olaquindox assisted by glucose oxidase-triggered Fenton reaction

ABSTRACTGlucose oxidase (GOx) catalyses oxidation of glucose accompanied with the generation of hydrogen peroxide. With the addition of Fe2+, hydroxyl radical produced by Fenton reaction between hydrogen peroxide and Fe2+ may quench the fluorescence of gold nanoclusters. In this work, a fluorescent enzyme-linked immunosorbent assay with gold nanoclusters was designed with a straightforward signal output, in which the fluorescence of gold nanoclusters was quenched by GOx-triggered Fenton reaction. Olaquindox was selected as a target analyte. Gold nanoclusters capped with bovine serum albumin and GOx-linked olaquindox conjugates were successfully prepared. Olaquindox in samples directly competed with the GOx-linked olaquindox conjugates for binding immobilized antibody. Consequently, the fluorescence signal increased with the amount of olaquindox. Under optimal conditions, the fluorescent enzyme-linked immunosorbent assay exhibited a favorable performance to detect olaquindox in swine feeds, demonstrating a good linear range from 1.0 µg kg−1 to 150 µg kg−1 with a reliable correlation coefficient (R2 = 0.9918); the limit of detection was 0.68 µg kg−1. Average recoveries in spiked samples were 85.3% to 113.5%. The proposed strategy is a promising approach for the detection of olaquindox and other harmful small molecules.

Potential Use of the Maillard Reaction for Pharmaceutical Applications: Gastric and Intestinal Controlled Release Alginate-Albumin Beads.

In this study, bovine serum albumin (BSA) and alginate (ALG) conjugates were synthesized by the Maillard reaction in order to evaluate their potential to develop controlled release drug delivery systems. The progress of the Maillard reaction was evidenced using ultraviolet (UV) absorbance, determination of BSA remaining free amino groups, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). BSA-ALG conjugates possessed enhanced and tunable viscosity, foamability and foam stability. Foam generated from BSA-ALG conjugate solution was used to prepare floating gastroretentive calcium ALG beads. Unlike traditional ALG beads, BSA-ALG foam beads were able to float and sustain the ciprofloxacin (CIP) release in gastric medium. Interestingly, intestinal beads made of ALG, BSA-ALG physical mixture and BSA-ALG conjugate resulted in different release rates and orders of indomethacin (IND) in simulated intestinal fluids; while beads based on a physical mixture of BSA-ALG resulted in a first order sustained release profile, both systems based on ALG and BSA-ALG conjugate displayed zero order sustained release profiles with IND being released at a slower rate from the conjugate beads.

Novel ractopamine-protein carrier conjugation and its application to the lateral flow strip test for ractopamine detection in animal feed.

In this work, a novel conjugate of ractopamine and bovine serum albumin (RAC-BSA) has been developed via the Mannich reaction, with a mole coupling ratio for RAC-BSA of 9:1. The proposed conjugation method provides a simple and one-step method with the use of fewer reagents compared with other conjugation methods for competitive immunoassays. RAC-BSA conjugation was used to fabricate a competitive lateral flow strip test for RAC detection in animal feed. For sample preparation, RAC was spiked in swine feed purchased from the local markets in Thailand, and methanol and running buffer at a volume ratio of 10:90 was used as extraction buffer. The procedures for sample preparation were completed within 25 min. Under optimal conditions, the limit of detection (LOD), assessed by the naked eye within 5 min, was found to be 1 ng/g. A semi-quantitative analysis was also conducted using a smart phone and computer software, with a linearity of 0.075-0.750 ng/g, calculated LOD of 0.10 ng/g, calculated limit of quantitation of 0.33 ng/g, and good correlation of 0.992. The recoveries were found in the range of 96.4%-103.7% with a relative standard deviation of 2.5%-3.6% for intra- and inter-assays. Comparison of the results obtained by the strip test with those obtained by enzyme-linked immunosorbent assay had a good agreement in terms of accuracy. Furthermore, this strip test exhibited highly specific RAC detection without cross reactivity with related compounds. Therefore, the RAC-BSA conjugation via the Mannich reaction can be accepted as a one-step and easy conjugation method and applied to the competitive lateral flow strip test.

Rapid and visual detection of benzothiostrobin residue in strawberry using quantum dot-based lateral flow test strip

A quantitative method was developed for the rapid, visual detection of benzothiostrobin residue in strawberry using the conjugates of CdSe/ZnS (core/shell) quantum dots (QDs) and anti-benzothiostrobin antibody (IgG) based fluorescence lateral flow test strip (FLFTS). The IgG with a binding specificity to the target antigen benzothiostrobin-BSA (a conjugate of benzothiostrobin and bovine serum albumin) was labeled with the QDs to prepare the QD-IgG bioprobes. The primary strategy included the designation of three test lines (T lines), which were sprayed with different concentrations of the antigen. After the spiked samples were added to the sample pad for 15 min at 37 °C, the results were visualized with the unaided eye with using ultraviolet (UV) protective goggles under UV lamp with a detection limit of 25 μg/L. Moreover, the visual detection method together with a gel imager, could be used as a quantitative method. The cut-off values for the T line 1, 2, and 3 were 1200, 600, and 150 μg/L, respectively. So multi-concentration and visual detection of spiked samples had been achieved. At last, the detection results of the spiked samples by FLFTS were consistent with the results of the UPLC-MS/MS method.

Interferometric detection of chloramphenicol via its immunochemical recognition at polymer-coated nano-corrugated surfaces

Three types of polymer-coated glass slides were prepared by acylation of their γ–aminopropyl derivatives with succinic anhydride or poly(p-nitrophenyl acylate) (PNPA) at various reaction times. The variations in duration of chemical adsorption of PNPA allowed control over nano-corrugation of the reactive surfaces employed for biosensing. The visually smooth and optically clear polymer-coated slides were used for immobilization of a conjugate of bovine serum albumin with chloramphenicol (CAP) and label-free measurement of concentration of this antibiotic. The sensor chips prepared in this way provided strong biosensing response due to fast biomolecular interactions and efficient competition of the target analyte during indirect immunoassay. The kinetics of formation and dissociation of immune complexes “conjugate-antibody” on the sensor chip surface was registered by the label-free methods of spectral-correlation and spectral-phase interferometry. The average thickness of immune complex adsorption layers was in the range of 5–12 nm. The highest value of kinetic association constant (kon = 5 × 104 M−1s−1) and the lowest equilibrium dissociation constant (Kdiss = 2 × 10-8 M−1) were achieved with the chips originated in the PNPA-coated slides, which were produced under short (2 min) polymer chemisorption and exhibited nano-corrugation almost unchanged with respect to the parent glass, according to the atomic-force microscopy. The detection limit for CAP detection was 80 pg/mL, and the dynamic range was 5 orders. The proposed methods for fabrication of sensor chips with nano-corrugated surface, the results of their characterization, as well as the developed assay can be employed for high-throughput recording of multiple molecular interactions by spectral interferometry and in a wide range of biosensing platforms for registration of biologically active compounds in medicine, veterinary, food control and ecological monitoring.

Adsorption of proteins on gold nanoparticles: One or more layers?

Adsorption of proteins on nanoparticles is a complex and poorly studied process. The mechanisms of protein layer formation can fundamentally differ depending on the composition of the medium, the nanoparticles' structure, the protein's nature, and other factors. In particular, monolayer or multilayer immobilization may occur. In the present work, the composition of conjugates of bovine serum albumin and immunoglobulin G with gold nanoparticles obtained by the Turkevich-Frens method are analyzed. The composition was studied by protein fluorescence measurement for particles ranging in size from 20 to 48 nm, depending on the pH of the immobilization medium (from 4 to 5 to 8-10). It was found that a pH shift of the immobilization medium from acidic to alkaline values is accompanied by a change in the mechanism of protein adsorption on the gold surface. In acidic pH conditions (4-5), effective binding of bovine serum albumin and gold nanoparticles occurs throughout the entire range of studied protein concentrations. In alkaline pH conditions (8-10), however, effective binding occurs only at concentrations of >10 μg/mL. This effect is not observed for immunoglobulin G, which is efficiently adsorbed onto nanoparticles throughout the entire range of studied concentrations and pH values. For acidic pH values, the surface of the particles is saturated with the amount of bound proteins, which approximately corresponds to the amount the monolayer is filled. For neutral and alkaline pH values, saturation is not observed and the amount of adsorbed protein certainly exceeds the monolayer filling, leading to multilayer immobilization.

Magnetic nanoparticles-based immunoassay for aflatoxin B1 using porous g-C3N4 nanosheets as fluorescence probes

With the porous g-C3N4 nanosheets (pg-CNNSs) as fluorescence probes and AFB1–bovine serum albumin conjugate modified Fe3O4 magnetic nanoparticles (MNPs) as immunosensing probes, a simple sensitive aflatoxin B1 (AFB1) detection method was first constructed. Based on the simple preparation method, the excellent biocompatibility, strong fluorescence emission and proper emission wavelength, pg-CNNSs were first functionalized by AFB1 antibody as fluorescence probes, which can bind with MNPs through antigen-antibody immunoreaction to form the pg-CNNSs-MNPs complexes. After magnetic separation, the fluorescence intensity of the complexes was tested. The target analyte (AFB1) can interfere the above immunoreaction by direct competition with immunosensing probes, and lead to the signal decreased. The detection limit for AFB1 was as low as 0.002 ng mL−1 under optimal conditions. Additionally, the applicability of this method was also confirmed in adulterated maize samples.

Gold-carbon nanoparticles mediated delivery of BSA: Remarkable robustness and hemocompatibility

There is a tremendous interest in the fabrication of novel hemocompatible nanoparticles for applications in theranostics. In this context, bovine serum albumin (BSA) conjugates with the water-soluble and super robust gold-aryl nanoparticles truncated with carboxylic functionals were synthesized. The gold-aryl nanoparticles were fabricated by the mild reduction of the diazonium gold(III) salt [HOOC-4-C6H4N≡N]AuCl4. The gold core size and the presence of BSA sheath encapsulating Au−COOH nanoparticles were concluded from transmission electron microscopy (TEM). Clear spectral changes supported by quantitative X-ray photoelectron spectrometry (XPS) surface analysis are visible: the C1s/Au4d intensity ratio increased sharply and the gold features were very well attenuated after BSA attachment, which brings more nitrogen essentially due to NH-C = O peptide links. The conjugation of BSA with Au−COOH nanoparticles resulted in the isoelectric point (pI) change from 4.7 to a range of 4.1-5.0. The robustness of the bioconjugates can be concluded based on the high ζ-potential values and the resistance to drastic pH conditions. The high encapsulation efficiency percentage (EE%) calculated using Bradford protein assay was in a range of 90.0–94.6% under physiological pH values. Taken together, overall studies imply the presence of interaction between the Au−COOH nanoparticles and BSA. In this study we investigated the interaction of gold nanoparticles and their BSA conjugates with human red blood cells (RBCs), which might cause cell deformity and hemolysis. To elaborate on the uniqueness of the bioconjugates, they showed negligible hemolysis or morphology deformation to cells membrane visualized using light and phase contrast microscopes. Quantitative measurements of hemoglobin leached out of the cell membrane using UV–vis showed negligible hemolysis. The high physiological stability of the conjugates fosters their applications in intravenous drug delivery supported by the long blood circulation half-life.

Improved chemical stability and cellular antioxidant activity of resveratrol in zein nanoparticle with bovine serum albumin-caffeic acid conjugate

In this study, bovine serum albumin (BSA)-caffeic acid (CA) conjugate was prepared with free radical-induced grafting method. The CA to BSA ratio of the conjugate was 115.7 mg/g. In vitro antioxidant activity assays suggested that BSA-CA conjugates had stronger antioxidant activity than BSA. Resveratrol-loaded zein encapsulated with BSA and BSA-CA conjugate core-shell nanoparticles were prepared with antisolvent method. Particle sizes were 206.3 nm, and 217.2 nm for BSA and BSA-CA, respectively. The encapsulation efficiencies (EEs) were 85.3% and 86.5% for zein-BSA and zein-BSA-CA nanoparticles, respectively. SEM results indicated that both nanoparticles were spherical with mean diameter approximately 200 nm and smooth surfaces. Both thermal and UV light stability of resveratrol was significantly improved after nanoencapsulation. BSA-CA conjugate showed remarkably greater protection than BSA against resveratrol degradation. Cellular antioxidant activity (CAA) study confirmed that resveratrol in both zein-BSA and zein-BSA-CA nanoparticles had significant higher antioxidant activities than resveratrol alone.

Gold nanoparticles as a factor of influence on doxorubicin–bovine serum albumin complex

The interaction between doxorubicin (Dox) and bovine serum albumin (BSA) complex with gold nanoparticles (AuNPs) was investigated by optical spectroscopy. The optical absorption of Dox and BSA solutions was studied. The formation of Dox–BSA complexes with a binding constant K = 7.56 × 106 M−2 and the number of binding sites n = 2 was found out. With pH 6.9, the concentration of complexes is an order of magnitude lower than the concentration of unbound antibiotic molecules. Optical absorption in solutions of Dox–BSA conjugates in the presence of AuNPs undergoes a significant rearrangement, which manifests the changes in the magnitude of the hydrophobic interaction of BSA with Dox, changes in the conformational state of antibiotic, and, as a consequence, a plasmon-induced change in the mechanism of complex formation. The aggregation of the Dox–AuNPs conjugate depends on the presence and concentration of BSA and in the case of formation of the Dox–BSA complex is minimal.

Thiodigalactoside-Bovine Serum Albumin Conjugates as High-Potency Inhibitors of Galectin-3: An Outstanding Example of Multivalent Presentation of Small Molecule Inhibitors.

Galectin inhibitors are urgently needed to understand the mode of action and druggability of different galectins, but potent and selective agents still evade researchers. Small-sized inhibitors based on thiodigalactoside (TDG) have shown their potential while modifications at their C3 position indicated a strategy to improve selectivity and potency. Considering the role of galectins as glycoprotein traffic police, involved in multivalent bridging interactions, we aimed to create multivalent versions of the potent TDG inhibitors. We herein present for the first time the multivalent attachment of a TDG derivative using bovine serum albumin (BSA) as the scaffold. An efficient synthetic method is presented to obtain a novel type of neoglycosylated proteins loaded with different numbers of TDG moieties. A polyethylene glycol (PEG)-spacer is introduced between the TDG and the protein scaffold maintaining appropriate accessibility for an adequate galectin interaction. The novel conjugates were evaluated in galectin binding and inhibition studies in vitro. The conjugate with a moderate density of 19 conjugated TDGs was identified as one of the most potent multivalent Gal-3 inhibitors so far, with a clear demonstration of the benefit of a multivalent ligand presentation. The described method may facilitate the development of specific galectin inhibitors and their application in biomedical research.

Sensitive quantitative analysis of the bitter glycoside amarogentin by specific monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay.

Amarogentin (AG) is a naturally occurring secoiridoid glycoside produced mainly in the plant genera Swertia and Gentiana. Extracts of these plants have a long history of use in Japan as bitter stomachics because of their strong bitterness. Because the AG content directly reflects the potential activity of the extract, the quality control of these plant extracts through the quantitative analysis of AG is important. In the present study, we aimed to develop a quantitative analysis of AG using a monoclonal antibody (MAb) against AG (MAb 1E9) in the plant family Gentianaceae. Surprisingly, production of MAb 1E9 was successfully achieved by immunizing AG–bovine serum albumin (BSA) conjugates into mice although the number of AG bound to BSA was only one. The characterization of MAb 1E9 revealed that it shows high specificity to AG, enabling the development of an icELISA for the specific determination of AG. In addition, the detectable concentration of AG in the developed icELISA ranged from 1.95 to 62.5 ng mL−1 with a limit of detection of 1.28 ng mL−1, which is approximately 30–625 times higher in sensitivity compared with the conventional HPLC method. Validation analysis revealed that the icELISA using MAb 1E9 is precise (intra-assay variation <3.9%, inter-assay variation <8.8%) and accurate (recovery rates of spiked samples were between 91.0% and 106.4%). This method can be used for the quality control of plant extracts using AG as an index.

Enzymatic catalyzed corn fiber gum-bovine serum albumin conjugates: Their interfacial adsorption behaviors in oil-in-water emulsions

The purpose of present study was to investigate the interfacial behaviors of the peroxidase-catalyzed intermolecular conjugates of corn fiber gum (CFG) and bovine serum albumin (BSA) (gum and protein weight ratio of 4:1). CFG-BSA conjugate stabilized emulsion remained stable with low flocculation and coalescence degree during storage in comparison to single BSA or CFG emulsion, attributing to its higher amount of adsorbed protein and polysaccharide. As shown by SDS-sulfate polyacrylamide gel electrophoresis, the adsorbed protein at the surface of oil droplets stabilized by CFG-BSA conjugate was partly crosslinked with CFG, forming an entangled emulsion network with increased apparent viscosity. Adsorbed CFG-BSA conjugates distributed loosely around the droplet interfaces as revealed by images of confocal laser scanning microscopy. Their interfacial layer thickness was estimated to be 85.1 nm using a monodisperse polystyrene latex particle model, which was not only larger than the adsorbed layer thickness of single CFG (71 nm) or BSA (6.9 nm) but also their combined layer thickness. To our knowledge, it is the first time that the interfacial properties of enzymatically-crosslinked polysaccharide and protein have been studied.

Efficient gene delivery by oligochitosan conjugated serum albumin: Facile synthesis, polyplex stability, and transfection

Chitosan and its derivatives have shown to be potential gene carriers with biocompatiblility and safety. However, their practical delivery is far from being ideal because of the low transfection efficiency. The present work describes the potential of a natural protein, bovine serum albumin (BSA), conjugated with a natural oligosaccharide, oligochitosan (OC), as a considerable promising approach for a safe and efficient non-viral gene delivery vector. The FTIR spectra proved the effective conjugation of BSA with OC through covalent bond. The condensation ability of plasmid DNA (pDNA) with a BSA-OC biopolymer was analyzed by gel retardation assay, competition binding assay, and dynamic light scattering used to measure the nanoparticle size. In addition, the BSA-OC biopolymer showed the protection of pDNA from enzymatic degradation by DNase I and showed good stability when exposed to 50% fetal bovine serum. The transfection efficiency was evaluated in the presence of 10% serum-supplemented media or serum-free media on three kinds of mammalian cells. Our results showed that the BSA-OC biopolymer is a good non-viral vehicle for gene delivery. We investigated the parameters such as the pDNA payload, temperature, incubating duration, and biopolymer/pDNA ratio on the transfection efficiency. This hybrid vehicle had the ability to transfect 90% of cells and to maintain 80% of cell viability. The aforementioned results suggest that the facile synthesis of the BSA-OC biopolymer could overcome the cytotoxicity problem and transfection barriers during in vitro gene delivery.