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PubMed2003

Enzyme stabilization strategies based on electrolytes and polyelectrolytes for biosensor applications.

The achievements in the area of enzyme stabilization based on electrolytes, polyelectrolytes and polyols is reviewed, in the context of biosensor applications. Both the storage and operational stabilities of the biosensors can be improved using these stabilizers. The deactivation of the enzymes used for the development of biosensors from thermal shock, proteolytic degradation, and non-specific metal-catalyzed oxidation can be drastically reduced with the use of one or more of these stabilizers. It is attempted to deconvolute the effect of these additives on (a) the storage stability or shelf life, and (b) the operational stabilities of the biosensors. Even though there are a large number of techniques and reports dealing with enzyme stabilization, their application to biosensor technology is still very limited. It is thus concluded that the use of the existing enzyme stabilization techniques will have a drastic effect on the storage and operational stabilities of biosensors in the near future.

Biosensing Techniques↗
PubMed2004

Ferrocene-containing polyelectrolyte multilayer film-covered electrodes: electrocatalytic determination of ascorbic acid and use of inner blocking layers to improve the upper detection limit of the electrodes.

A multilayer film composed of ferrocene(Fc)-appended poly(allylamine hydrochloride) (Fc-PAH) and poly(potassium vinylsulfate) (PVS) has been prepared on the surface of a gold(Au) electrode by using a layer-by-layer self-assembly technique. Fc-containing polyelectrolyte multilayer (PEM) film-modified electrodes can electrochemically catalyze the oxidation of ascorbic acid successfully. For a 2 (Fc-PAH/PVS) bilayer-covered electrode the catalytic current increased linearly with increasing concentration of ascorbic acid over the concentration range 6 micromol L(-1)-3 mmol L(-1). To extend the dynamic range for ascorbic acid, the surface of the Au electrode was first covered with a (PAH/PVS)(2) film on which an additional (Fc-PAH/PVS)(5) film was coated. This strategy successfully extended the dynamic range of the electrode up to 25 mmol L(-1) ascorbic acid, because the (PAH/PVS)(2) layer blocked access of ascorbic acid to the electrode surface. The upper detection limit of the (PAH/PVS)(2) (Fc-PAH/PVS)(5) film-modified electrode is much higher than those of Fc-based ascorbic acid sensors reported so far. Electron transfer is diffusion-controlled within the (PAH/PVS)(2)(Fc-PAH/PVS)(5) film.

Ascorbic Acid↗
PubMed2005

The selectivity in MEKC of pseudo-stationary phases based on polyelectrolyte complexes: I. Composition of the complex.

Polyelectrolyte-surfactant complexes (PSC) of polycarboxylic acids with alkyl-trimethylammonium salts look very promising as a new type of pseudo-stationary phase in micellar electrokinetic chromatography. PSC produce an intramolecular micellar phase, and the morphology of the micelles is significantly different from that of the corresponding "typical" surfactant micelles. Pseudo-stationary phases based on PSC have unique selectivity. In this paper, the effect of the composition (phi) of the PSC of polyacrylic acid (PAA) M(W) 130,000 with dodecyltrimethylammonium bromide (DTAB) and of the PSC of PAA M(W) 450,000 with DTAB on the separation of DNS-amino acids and phenol derivatives in these systems was investigated. Relative retention and relative selectivity were used to describe the electrophoretic behavior of the amino acids and phenol derivatives. The main advantage of PSC pseudo-phases is that the nature and the structure of micelle-like units, and hence the selectivity of electrophoretic separation, could easily be modified by changing the composition of the complex.

Journal Article↗
PubMed2006

Polyelectrolyte complex formation mediated immobilization of chitosan-invertase neoglycoconjugate on pectin-coated chitin.

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on pectin-coated chitin support via polyelectrolyte complex formation. The yield of immobilized enzyme protein was determined as 85% and the immobilized biocatalyst retained 97% of the initial chitosan-invertase activity. The optimum temperature for invertase was increased by 10 degrees C and its thermostability was enhanced by about 10 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 4-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The biocatalyst prepared retained 96 and 95% of the original catalytic activity after ten cycles of reuse and 74 h of continuous operational regime in a packed bed reactor, respectively.

Bioreactors↗
PubMed1986

Conformations of hydrophobic polyelectrolytes.

Recent studies on the (hypercoiled) compact conformation and the pH-induced conformational transition to the extended coil of the hydrophobic poly(acids or bases), which have been well used in industrial and medical circles, are summarized, especially on the basis of our studies on poly(alpha-alkyl acrylic acids) and maleic acid copolymers with hydrophobic aromatic monomers. The stability of the compact conformation and values of various thermodynamic parameters of the transition of the hydrophobic polyelectrolytes suggest the importance of hydrophobic interaction among the side chains to form the compact conformation. Characterization of various physicochemical methods, including 1H-NMR measurements, gives a model of the compact conformation with a more expanded structure than in globular proteins, a free-draining property of the solvent, the conformation fluctuation, etc. Also, the apparent two-state character of each segment in the transition region, a life-time in each state and the cooperative parameter for the compact form formation are discussed. A review of our conformational studies on the hydrophobic-hydrophilic polypeptides is also given. For the anion-induced conformation transition in basic homopoly(alpha-amino acids), hydrophobic characters of the anion and the side chain in the polypeptide are shown to be important, especially in terms of solvation. The difference in the induced conformation by the anion between random and alternating basic copolypeptides is explained by introducing a model which shows the importance of the sequence of hydrophobic and hydrophilic residues in the polypeptide to induce the ordered conformation of the anions. Also, we attempt to explain the difference between the induction of ordered conformation in the basic homo- or copolypeptide in reversed micelles with a large sulfonate (AOT) vs. that in aqueous AOT in terms of the hydrophobic character of the side chains in the polypeptides, AOT and the medium.

Electrolytes↗
PubMed1996

Kinetics of membrane micellization by the hydrophobic polyelectrolyte poly(2-ethylacrylic acid).

Rates of pH-dependent micellization of multilamellar vesicles by the hydrophobic polyelectrolyte poly(2-ethylacrylic acid) (PEAA) have been measured turbidometrically. This polymer shows a strong ph-dependence in its affinity for phospholipid membranes, binding in increasing amounts as pH is lowered and ultimately solubilizing membranes to form mixed micelles (Tirrell, Takigawa and Seki (1985) Ann. N.Y. Acad. Sci. 446, 237). The rate of solubilization of dipalmitoylphosphatidylcholine (DPPC) vesicle suspensions by PEAA increases approximately linearly with reductions in pH below a threshold at pH 6.55. Interestingly, negatively-charged dipalmitoylphosphatidylglycerol membranes showed qualitatively similar behavior in the presence of PEAA, and incorporation of 10% or 20% dipalmitoylphosphatidic acid in DPPC membranes did not affect solubilization rates, demonstrating that membrane charge is not an important factor in determining micellization kinetics. Micellization of DPPC and dimyristoylphosphatidylcholine membranes occurs most rapidly at their respective gel-liquid crystalline transition temperatures (Tm); the rate enhancement is correlated with a peak in the temperature-dependent binding of a fluorescently-modified PEAA in slightly alkaline solutions in which no micellization is observed. The lateral compressibility of the membrane, which has a similar peak at Tm, is proposed to be an important determinant of the rate and extent of polymer adsorption, and consequently of the rate of micellization.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1989

The use of polyelectrolyte-fractionated porcine factor VIII in the treatment of a spontaneously acquired inhibitor to factor VIII.

Polyelectrolyte-fractionated porcine factor VIII concentrate is a recent addition to the therapeutic choices for treatment of factor VIII inhibitor patients, but cross-reactivity of the inhibitor with porcine factor VIII limits its usefulness in some cases. Hemophilic patients with inhibitor titers greater than or equal to 50 Bethesda units/micromilligrams often demonstrate sufficient cross-reactivity (10-20%) to prevent the achievement of a satisfactory plasma factor VIII level and a therapeutic response with porcine factor VIII. We have studied plasma from five women with high-titer, spontaneously acquired factor VIII inhibitors to determine the degree of cross-reactivity with porcine factor VIII. Four of the five had little or no detectable inhibitor to porcine factor VIII despite high titers to human factor VIII (26-143 Bethesda units/micromilligrams). One of these patients, with a titer of 53 Bethesda units/micromilligrams against human factor VIII, was treated successfully with porcine factor VIII concentrate, given for serious hemorrhagic complications. These studies and other reports support the conclusion that the majority of high-titer spontaneous factor VIII inhibitors exhibit little cross-reactivity with porcine factor VIII and can be treated successfully with this product.

Adult↗
PubMed1989

Polyelectrolytic effects in semi-flexible carboxylate polysaccharides. Part 2.

A statistical model is presented in which an ionic polymer is taken as a linear arrangement of flexible segments (similar to the spring-bead model). This model is used to represent semi-flexible linear ionic polysaccharides in solution. The distribution of end-to-end distances is taken from Monte Carlo calculations of the amylosic chain conformation and combined with Manning's counterion condensation theory of linear polyelectrolytes. The excess thermodynamic properties are then calculated as a function of the degree of ionization and of a number of physical variables. The results show that the statistical average of thermodynamic functions taken over the conformational states is not equivalent to the thermodynamic function of the average conformation. This has important implications for the correct comparison of theoretical prediction with the experimental results.

Carbohydrate Conformation↗
PubMed1990

Polyelectrolyte and rheological studies on the polysaccharide welan.

This paper investigates the polyelectrolyte behaviour of welan in aqueous solution. From conductivity and activity measurements it is demonstrated that a double-helical conformation is adopted irrespective of pH and ionic strength. The intrinsic pK of its carboxylic groups is found to be abnormally low (pKO = 2.2) and no conformational transition was observed during neutralization. The viscometric behaviour was studied as a function of degree of neutralization, polymer concentration, shear rate and ionic strength. The linear relation [eta] = f(Cs-1/2) allows the determination of B, the stiffness parameter from Smidsrød, whose value characterizes a very stiff molecule.

Carbohydrate Conformation↗
PubMed1982

Dental polyelectrolyte cements. I. Chemistry of the early stages of the setting reaction.

As part of an investigation of the setting of dental polyelectrolyte cements, the chemistry of a selection of glass ionomer and zinc polycarboxylate cements was studied by pH, conductivity and I.R. measurements. The zinc polycarboxylate cements were found to react at a greater rate than the glass ionomer cements. The effect of reducing the powder/liquid (P/L) ratio is to decrease the surface area available to attack and hence the reaction rate. The basic form of the kinetics appears to be unaffected except for low P/L ratios where there is a deficiency of available metal cations.

Chemical Phenomena↗
PubMed1983

Dental polyelectrolyte cements. III. Effect of additives on their rheology.

As part of a series of investigations of dental polyelectrolyte cements, the influence of additives was studied by viscometry. At low concentrations, tartaric acid was found to exert an accelerating influence on glass ionomer cements but to retard the reaction by complex formation at high concentrations. When dissolved with the liquid component, NaF accelerated some glass ionomer cements and retarded others. NaCl was found to accelerate the zinc polycarboxylate cements, possibly through its reduction in the electrical potential between powder and liquid phases. Neutralization of the polyacids retarded the reactions but not enough to indicate that hydrogen ions were directly involved in the rate determining step.

Dental Cements↗
PubMed1983

Dental polyelectrolyte cements: II: effect of powder/liquid ratio on their rheology.

The rheological behaviour during the setting of a range of zinc polycarboxylate and glass ionomer dental cements has been studied. The influence of the powder/liquid ratio was found to alter the rate of reaction without altering the basic form of the kinetics. Two models were advanced to explain the rheological and chemical differences between the two types of polyelectrolyte cement. The setting of the glass ionomer cements was consistent with the development of a homogeneous polymer network whereas the zinc polycarboxylate cements were viewed as setting by an inhomogeneous core-growth reaction.

Dental Cements↗
PubMed1996

Encapsulation of artificial tissues in polyelectrolyte complexes: preliminary studies.

The in vitro engineering of vital tissues from isolated cells requires primarily the synthesis of a new intercellular matrix. Structural components of the extracellular matrix are large molecules such as collagens and proteoglycans. To retain and accumulate new matrix molecules within three-dimensional cell cultures, chondrocyte-polymer constructs were encapsulated in polyelectrolyte complex membranes. Further, these membranes might also be relevant for other applications where cells or tissues have to be isolated from their environment by semipermeable structures.

Cells, Cultured↗
PubMed1994

A new visual enzyme immunoassay of methamphetamine using linear water-soluble polyelectrolytes.

A new visual enzyme immunoassay (EIA) technique has been developed. Oppositely charged synthetic linear water-soluble polyelectrolytes (poly-N-ethyl-4-vinyl-pyridine as polycation and polymethacrylate as polyanion) were used as carriers for reagent immobilization. The ability of these molecules to form an insoluble complex was applied for the separation of bound and free components of the immunoassay reaction mixture. This approach was realized in methamphetamine visual EIA. In the first stage of the assay two specific reactions took place during incubation of the analytical reagents with the probe to be analyzed: (1) competition between methamphetamine and hapten conjugated with peroxidase for the interaction with specific antibodies and (2) interaction of these antibodies with the protein A-polymethacrylate conjugate. As a result of these reactions the (polyanion-protein A)-antibody-(hapten-peroxidase) complex was formed. Then the reaction mixture was filtered through an Ultrabind membrane (0.45 microns) with adsorbed poly-N-ethyl-4-vinylpyridine, and the immunological complexes were immobilized to the membrane by electrostatic interaction. The level of peroxidase binding on the membrane was measured by diaminobenzidine substrate. The system described was optimized to achieve both high rapidity (20 min) and an appropriate sensitivity (0.4 micrograms/ml) for methamphetamine assay.

Animals↗
PubMed1990

Polyelectrolyte precipitation of beta-galactosidase fusions containing poly-aspartic acid tails.

Protein recovery from industrial microbial processes can be very expensive, often exceeding the cost of protein production. We have genetically engineered 3 beta-galactosidase (beta-gal) fusion proteins containing poly-aspartic acid tails to test the effect of the tails on recovery by the relatively inexpensive method of polyelectrolyte precipitation. The fusion proteins, designated T1, T2, and T3, were constructed with C-terminal tails of 5, 11, and 16 aspartic acid residues, respectively. The fusion proteins were expressed in Escherichia coli, and purified by affinity chromatography. T1 and T2 had specific activities similar to that of wildtype beta-gal, whereas the specific activity of T3 was about half that of T1 and T2. The increased net charge of the fusion proteins compared to wildtype beta-gal was indicated both by ion-exchange chromatography and their migration pattern in non-denaturing polyacrylamide gel electrophoresis. All three tails enhanced polyethyleneimine (PEI) precipitation of the fusion proteins compared to wildtype beta-gal. At a low PEI/protein ratio (0.01, g g-1), recovery by precipitation of T2 and T3 was more than 2 X that of the beta-gal control, whereas that of T1 was only slightly greater than that of the control. At a higher PEI/protein ratio (0.03, g g-1) the amount of precipitation of all three fusion proteins was nearly the same, about 1.5 X that of the control.

Aspartic Acid↗
PubMed1985

Influenza virus antigens conjugated with a synthetic polyelectrolyte: a novel model of vaccines.

Haemagglutinin (HA), a mixture of haemagglutinin and neuraminidase (HA + NA), and matrix (M) protein were isolated from the influenza A virus and covalently coupled to a synthetic polyelectrolyte (P). A single injection into mice of the resultant conjugates (virogates) brought about efficient stimulation of the primary immune response specific to the corresponding viral antigens. Mice immunized with virogates HA.P or (HA + NA).P were largely protected against a lethal challenge infection with homologous virus. Immunization of mice with M.P virogate containing M protein originated from a 1934 influenza strain resulted in pronounced protection against a lethal challenge infection with a 1980 strain. Virogates are discussed as a novel model of artificial vaccines.

Acrylic Resins↗
PubMed1975

Examination of the limiting laws of polyelectrolytes and counterion condensation II.

The two phase model of polyelectrolyte solutions, which has been developed recently, is examined in a further detail. The binding free energy, which was introduced in the previous paper (J. Chem. Phys. 81 (1977) 1929), is replaced by the entropy of the condensed phase. This replacement leads to a detailed picture of the condensed phase. In a mixed system of mono- and divalent counterions, a couple of possibilities are examined in interpreting the condensation volume, which corresponds to the condensation entropy.

Journal Article↗
PubMed1976

Magnetic resonance distinction between site bound and atmospherically bound paramagnetic counterions in polyelectrolyte solutions.

A combination of the water protons NMR chemical shifts, longitudinal and transversal relaxation rates and of the paramagnetic counterion EPR signal is shown to provide a clear distinction between site binding, atmospheric trapping and free counterions in solutions of polyelectrolyte TMA salts with increasing concentrations of the divalent counterions Co++ and Mn++. Site binding is defined by the loss of water in the counterion first hydration shell while atmospheric binding results in a change in the counterion correlation time as compared to a free ion.

Binding Sites↗