Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.     

Changes in the refractive index of a solution during proteolysis of bovine serum albumin with pepsin

The protein refractive index was observed to increase by 8 · 10^–6 during proteolysis of bovine serum albumin (BSA, 4 mg/mL) with pepsin (2.2 mg/mL). It was believed earlier that the refractive index of a protein solution depends only on the protein concentration and amino-acid composition and remains unchanged during protein hydrolysis. An increase in the refractive index during proteolysis in solution was detected using an original laser interferometer with an improved sensitivity. The interferometer makes it possible to measure the difference in refractive index between solutions in two cells to an accuracy of approximately 6 · 10^–7.     

Maleimide-mediated protein conjugates of a nucleoside triphosphate gamma-S and an internucleotide phosphorothioate diester.

The purpose of this study was to determine whether the gamma-S of nucleoside thiotriphosphates and the non-bridging sulfur of internucleotide phosphorothioate diesters possess sufficient thiol character to form adducts with maleimides. Adenosine triphosphate gamma-S (ATPS) and thymidyl-PS-thymidine (TPST) were each reacted with the reporter molecule N-1 pyrene maleimide (PM) and the fluorescence intensity was recorded. The observed reactivity of the phosphorothioate nucleotides towards maleimide was used as a basis for preparing covalent protein-nucleotide conjugates of ATPS and of the internucleotide phosphorothioate diester, deoxyadenylyl-PS-deoxy-adenylyl-PS-deoxyadenosine (dA3(PS)2). The absorbance spectra of bovine serum albumin (BSA) conjugates of ATPS and of dA3(PS)2 showed the formation of protein-nucleotide conjugates, with absorbance maxima near 260 nm. The degree of conjugation was 1.69 nucleotides (nt)/BSA molecule for ATPS and 0.44 nt/BSA molecule for dA3(PS)2. The extent of conjugation of the gamma-S of the nucleoside thiotriphosphate and of the non-bridging sulfur of the internucleotide phosphorothioate diester with maleimide-derivatized protein agreed with their relative reactivity towards PM. Both the gamma-S of the nucleoside thiotriphosphate and the internucleotide phosphorothioate diester were found to possess sufficient thiol character to permit formation of maleimide-mediated protein conjugates.     

Preparation and recycling of aqueous two‐phase systems with pH‐sensitive amphiphilic terpolymer PADB

In this study, a novel pH‐sensitive terpolymer PADB was synthesized by random terpolymerization of 2‐(dimethylamino) ethyl methacrylate, acrylic acid, and butyl methacrylate. The terpolymer PADB could form aqueous two‐phase systems (ATPS) with a light‐sensitive terpolymer PNBC, which was synthesized in our laboratory, using n‐isopropylacrylamide, n‐butyl acrylate, chlorophyllin sodium copper salt as monomers. More than 97% of the PADB terpolymer could be recovered by adjusting the pH to isoelectric point (PI) 4.1. The terpolymer PNBC could be recovered by using light radiation at 488 nm, with recovery ratio of 98%. BSA and lysozyme were partitioned in the PNBC–PADB ATPS to examine this new system. It was found that the partition coefficient of BSA and lysozyme could reach 4.46 and 0.49 in the systems, respectively. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Effect of salt additives on protein partition in polyethylene glycol–sodium sulfate aqueous two-phase systems

Partitioning of 15 proteins in polyethylene glycol (PEG)–sodium sulfate aqueous two-phase systems (ATPS) formed by PEG of two different molecular weights, PEG-600 and PEG-8000 in the presence of different buffers at pH 7.4 was studied. The effect of two salt additives (NaCl and NaSCN) on the protein partition behavior was examined. The salt effects on protein partitioning were analyzed by using the Collander solvent regression relationship between the proteins partition coefficients in ATPS with and without salt additives. The results obtained show that the concentration of buffer as well as the presence and concentration of salt additives affects the protein partition behavior. Analysis of ATPS in terms of the differences between the relative hydrophobicity and electrostatic properties of the phases does not explain the protein partition behavior. The differences between protein partitioning in PEG-600–salt and PEG-8000–salt ATPS cannot be explained by the protein size or polymer excluded volume effect. It is suggested that the protein–ion and protein–solvent interactions in the phases of ATPS are primarily important for protein partitioning.     

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.     

The Effect of Bumpy Structure on Optical Properties of Bimetallic Nanoshells

In this paper, a sensitive bumpy bimetallic nanoshell for detection of thyroid cancer market (Thyroglobulin, Tg) and bovine serum albumin (BSA) proteins is reported. The physical origin of plasmonic properties of bimetal nanoshells is described by plasmon hybridization theory which indicates three intense and clearly separated plasmon modes. The electric field intensity enhancement of the bumpy bimetal nanoshell increases by ~559 %, at the surface of the bump in comparison with a smooth shell. The presence of bumpy structure on the nanoshell surface provides a high enhancement of the resulting Raman signal through an electromagnetic field of the order of 10⁷ which leads to an increase in sensitivity detection of Tg and BSA proteins. In addition, a refractive index (RI) sensitivity of 332.54 nm/RIU is achieved for this bumpy bimetallic nanoshell.     

Evaluation of polyvinyl alcohol for fatty acid supplementation in adipose tissue explant culture

Cultures of adipose tissue explants are a valuable tool for studying the intracellular mechanisms involving hormones and nutrients. However, testing how fatty acids affect cells requires a carrier molecule; bovine serum albumin (BSA) has been used for this purpose. However, contaminants can alter the cellular response. Our objectives were to: 1) test BSA as a fatty acid carrier and 2) evaluate polyvinyl alcohol (PVA) as a replacement for BSA. Adipose tissue explants from nine pigs were cultured in medium 199 for 4, 12, 24, and 48 h, with the following treatments: control, PVA (100 mM PVA added) and PVA + pGH (100 mM PVA plus 0.1 mg/mL porcine growth hormone). After each culture period, explants were collected and assayed for lipogenesis. After 48 h in culture, explants were assayed for lipolysis. A preliminary study with different commercial sources and high concentrations showed that BSA affected lipogenic rates. On the other hand, there were no effects of PVA on lipid synthesis, while pGH (positive control) reduced glucose incorporation into lipids (P < 0.01) when compared to both control and PVA (P < 0.05). There was no difference between control and PVA for lipolysis rates. However, pGH increased lipolysis when compared to control (P < 0.01) and PVA (P < 0.05). We demonstrated that BSA can alter lipogenesis, which precludes its use as a carrier molecule. On the other hand, addition of PVA had no effect on lipolysis or lipogenesis. We suggest the use of PVA instead of BSA for adding bioactive fatty acids to cultures of adipose tissue.     

Use of chemically modified proteins to study the effect of a single protein property on partitioning in aqueous two-phase systems: Effect of surface hydrophobicity

Two different series of hydrophobically modified proteins were partitioned in a number of aqueous two-phase systems (ATPS) to investigate the effect of hydrophobicity as a single property on partitioning. The modified proteins were derived from beta-lactoglobulin and bovine serum albumin (BSA). Measurement of the surface hydrophobicity of the proteins is important; hydrophobic interaction chromatography (HIC) was used for this purpose. The resolution of the systems (R) in terms of protein surface hydrophobicity and the intrinsic hydrophobicity (log P(0)) of the systems was established. The effect of the addition of NaCl to PEG/phosphate and PEG/dextran systems was analyzed in terms of the hydrophobicity difference between the phases and their ability to promote hydrophobic interactions between the protein surface and the PEG molecules. The values for R and log P(0) differed somewhat depending on which group of modified proteins was used for partitioning. The addition of NaCl to PEG/phosphate systems promoted an increase in the values of R, showing an important effect on the resolution of the systems for protein surface hydrophobicity (twice as high when compared with systems without NaCl). For PEG/dextran systems, the addition of 9% NaCl (w/w) promoted an improvement in the resolution toward surface hydrophobicity with an increase of 60% on the value of R. (c) 1996 John Wiley & Sons, Inc.     

Resonance of Different Waveguide Structures with Various Vertical Indirect Coupled Cavities

In the paper, resonances of different waveguide structures with various vertical indirect coupled cavities were investigated by FDTD (finite difference-time domain). In the silicon cavity, Fano resonance could be observed at about 1430 nm. The coupling distance for the gold cavity/air cavity had less effect on the transmittance of the main waveguide but had a great influence on the transmission for water cavity in the visible region, which showed that water cavity could adjust resonance of waveguide structures. In addition, with the increment of refractive index n, the resonance peak at about 850 nm moved to the long wavelength (redshift). Dispersion rate about 2 × 10^–3/nm indicated that the transparent dielectric selectively absorbed the surface plasmon polariton wave and the sensitivity of the waveguide structure designed in this paper has high stability for the refractive index of the main waveguide cavity. Obvious Fano resonance could be observed with the increase of refractive index for silicon cavity. Among the four dielectrics, silicon and water are suitable for studying Fano resonance and filter dielectrics.

     

The meiotic response of cumulus cell-enclosed mouse oocytes to follicle-stimulating hormone in the presence of different macromolecules.

Experiments were carried out to determine the effect of different macromolecules on the follicle-stimulating hormone (FSH)-induced maturation of mouse oocytes in culture. Cumulus cell-enclosed oocytes (CEO) were isolated from gonadotropin-primed mice and maintained in meiotic arrest for 17-18 h with the cAMP analogue, dibutyryl cAMP (dbcAMP). Germinal vesicle breakdown (GVB) was stimulated by the addition of FSH. Medium was supplemented with either no macromolecule or with varying concentrations of polyvinylpyrrolidone (PVP), polyvinylalcohol (PVA), crystallized bovine serum albumin (BSA), or fetal bovine serum (FBS). Oocyte maturation in all FSH-free cultures occurred at a frequency of about 30% or below. High frequencies of maturation were achieved when FSH was added to macromolecule-free medium or to cultures containing PVP, PVA, or BSA. Crystallized BSA was the most effective of these in supporting stimulation of maturation (94% GVB at 3 mg/ml, compared with 72-74% with synthetic polymer-supplemented or macromolecule-free media). The BSA effect was not due to contaminating fatty acids, and a less pure fraction V BSA was not as effective in supporting FSH-induced maturation. FBS suppressed FSH stimulation of maturation in a dose-dependent fashion. Sera from pigs, goats, horses, and rats were also inhibitory, but bovine calf serum (BCS) permitted a high maturation frequency (80% GVB). When added to medium containing either FBS or BCS, crystallized BSA had no effect on FSH-stimulated maturation, but fraction V BSA suppressed maturation in both serum-supplemented media. Under no conditions did FSH stimulate maturation in cumulus cell-free oocytes. These results demonstrate that hormone-induced oocyte maturation is supported in vitro by nonprotein polymers as well as BSA and that the behavior of the oocyte-cumulus cell complex depends on the purity of the BSA sample. In addition, serum contains inhibitory factors that suppress the positive response to FSH. Thus, the choice of macromolecular supplement is of critical importance when testing the hormone responsiveness of isolated cumulus cell-enclosed oocytes in culture.     

利用表面等离子体共振仪检测黄瓜花叶病毒

目的：研究一种便捷、高效地检测黄瓜花叶病毒（CMV）的方法。方法：利用表面等离子体共振（SPR）技术检测CMV。首先用11-MUA修饰SPR金片,再用EDC／NHS活化,之后通过NHS酯基与CMV抗体结合,用BSA封闭未结合的NHS酯基。将SPR金片装入SPR仪,通入待检样品,通过折射率变化实时监测实验过程。结果：该方法检测CMV的灵敏度能够达到10ng／mL,具有良好的特异性,与同属的花生矮化病毒、番茄不孕病毒无交叉反应。结论：建立的SPR方法操作简单、灵敏度高、特异性好,是一种新的高效检测CMV的方法。     

Ölgehalt und Sättigungsgrad der Öle sowie korrelative Beziehungen zwischen einigen Merkmalen bei Leinmutanten

Summary An analysis of the oil content and the refractive index (as a measure of the saturation value of the oil) was carried out during 1961–1964 in a collection of about 700 mutants, derived by X-irradiation from the two stocks no. 6 and no. 36.The average refractive index of the oil was highest under the cool and rainy conditions of 1961, lowest in the warm and dry year 1963. The maximum differences in the mean refractive index of the collections between 1961 and 1963 amount to an iodine number of 6 (mutants of stock no. 6) or 5 (mutants of stock no. 36) resp.The variability of both characters mentioned was significantly increased in the two mutant stocks. The average oil content showed, on the other hand, a decrease of about 1%. The degree of unsaturation of the oil is higher in the mutants derived from no. 6, and lower in the mutants derived from no. 36 when compared with the untreated material. In the present investigation no (no. 36) or negative correlation (no. 6) was found for the characters of oil content and refractive index.Subgrouping of such characters as plant height, seed coat color, flower color, etc. lead to more or less considerable differences in oil content and refractive index. It may be pointed out that mutants with yellow seed coat color are always characterized by a high oil content and a high refractive index.     

Detection of protein orientation on the silica microsphere surface using transverse electric/transverse magnetic whispering gallery modes

The state of adsorbed protein molecules can be examined by comparing the shifts in a narrow line resonance wavelength of transverse electric (TE) and transverse magnetic (TM) whispering gallery modes (WGM) when the molecules adsorb onto a transparent microsphere that houses WGM. In adsorption of bovine serum albumin (BSA) onto an aminopropyl-modified silica microsphere, the TM/TE shift ratio indicated highly anisotropic polarizability of BSA in the direction normal to the surface, most likely ascribed to anchoring the heart-shaped protein molecule by one of its tips. The polarization-dependent resonance shift was confirmed when the surrounding refractive index was uniformly changed by adding salt, which would simulate adsorption of large objects.     

Development, molecular composition and freeze tolerance of bovine embryos cultured in TCM-199 supplemented with hyaluronan

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p<0.05). Following freeze/thawing, significantly more (p<0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p<0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p<0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.     

Hypothermic storage of sheep embryos with antifreeze proteins: development in vitro and in vivo

Antifreeze proteins (AFPs) non-colligatively lower the freezing point of aqueous solutions, block membrane ion channels and thereby confer a degree of protection during cooling. Ovine embryos following prolonged hypothermic storage were used to determine 1) the type and concentration of a group of AFPs that can confer hypothermic tolerance, 2) the storage temperature, 3) the cooling rate, and 4) the in vitro and in vivo viability. In Experiment 1, Grade 1 and 2 embryos produced following superovulation were either cultured fresh (control) or stored at 4 degrees C for 4 d in media containing protein from 1 of 3 sources: Winter Flounder (WF; AFP Type 1); Ocean Pout (OP; AFP Type 3) at a concentration of 1 or 10 mg/ml; or bovine serum albumen (BSA) at 4 mg/ml in phosphate buffered saline (PBS). Following 72 h of culture, the viability rates were not different between controls (18 21 ); BSA (9 15 ); WF at 1 mg/ml (14 15 ); WF at 10 mg/ml (13 15 ) or OP at I mg/n-d (15 21 ), but were decreased (P < 0.05) in embryos stored in OP at 1 0 mg/ml (I 1 20 ). Pooled data showed higher (P < 0.05) viability rates for WF (27 30 ) than for OP (26 41 ) or BSA (9 15 ). There was no effect of protein source on hatching rates, but mean hatched diameters of embryos were lower (P < 0.05) following storage in BSA. In Experiment 2, Grade I to 3 embryos were either cultured fresh or stored for 4 d at 0 degrees or 4 degrees C in 4 mg/n-d BSA or 1 mg/ml WF. Embryos stored in WF at 4 degrees C (WF/4 degrees C) had comparable hatching rates (8 12 ) to that of controls (10 10 ), but embryos in the other treatments (WF 0 degrees C, 5 11 , BSA 4 degrees C, 6 11 and BSA 0 degrees C, 3 10 ) had significantly lower hatching rates (P < 0.01) compared with controls. Hatched diameters were comparable between controls and embryos stored in WF 4 degrees C, but embryos stored in WF 0 degrees C and BSA at both temperatures had smaller diameters (P < 0.05). In Experiment 3, Grade 1 to 3 embryos were either transferred fresh or were stored for 4 d at 4 degrees C in 4 mg/ml BSA or 1 mg/ml WF at different cooling rates (T1, BSA > 2 degrees C/min; T2, WF > 2 degrees C/min and T3, WF < 1 degrees C/min) prior to transfer. There were no differences in the number of ewes pregnant (T1, 10 1 1; T2, 6 10 and T3, 8 10 ) or in the number of viable fetuses recovered per treatment (T1, 14 25 ; T2, 10 1 4 and T3, 15 2 1) to indicate a negative effect of cooling rate or protein on embryo survival. In conclusion, ovine embryos can be stored in WF or BSA at 4 degrees C for 4 d, yielding similar pregnancy and embryo survival rates as fresh embryos following transfer to recipient ewes.     

Highly Sensitive Surface Plasmon Resonance Based D-Shaped Photonic Crystal Fiber Refractive Index Sensor

In this article, a D-shaped photonic crystal fiber based surface plasmon resonance sensor is proposed for refractive index sensing. Surface plasmon resonance effect between surface plasmon polariton modes and fiber core modes of the designed D-shaped photonic crystal fiber is used to measure the refractive index of the analyte. By using finite element method, the sensing properties of the proposed sensor are investigated, and a very high average sensitivity of 7700 nm/RIU with the resolution of 1.30 × 10^?5 RIU is obtained for the analyte of different refractive indices varies from 1.43 to 1.46. In the proposed sensor, the analyte and coating of gold are placed on the plane surface of the photonic crystal fiber, hence there is no necessity of the filling of voids, thus it is gentle to apply and easy to use.     

Enhancement of antibody production by growth factor addition in perfusion and hollow-fiber culture systems

The effects of the high-molecular-weight growth factors, transferrin and bovine serum albumin (BSA), on antibody production were analyzed quantitatively in continuous hollow-fiber cultivation over a period of 60 days. Transferrin enhanced cell growth but had no significant effect on the specific antibody production rate, whereas BSA significantly enhanced antibody production. The antibody production rate was increased 4- and 14-fold respectively by feeding BSA at 2 and 5 g L(-1) into the EC side of the system (the side connected to the cell-containing outer part of the hollow-fiber unit) compared with the production achieved without BSA. Addition of 5 g L(1) BSA into the IC side of the system (the side connected to the inner part of the hollow-fiber unit) resulted in a 2.5-fold increase in the antibody production rate. The effect of BSA was also analyzed using the perfusion culture system with a separation unit. When fresh medium containing either 2 or 5 g L(-1) BSA was fed into the reactor, both the specific growth rate and specific death rate increased, while the specific antibody production rate was increased 2- and 25-fold, respectively, by feeding BSA at these two concentrations compared with no addition. Comparing the two systems, the increase in the antibody production rate achieved with the hollow-fiber system was threefold greater than that in the perfusion culture system with the same concentration of BSA feeding. (c) 1995 John Wiley & Sons, Inc.