Immobilization of Invertase in a Novel Proton Conducting Poly(vinylphosphonic acid) – poly(1-vinylimidazole) Network

A novel proton conducting polymer blend was prepared by mixing poly(vinylphosphonic acid) (PVPA) with poly(1-vinylimidazole) (PVI) at various stoichiometric ratios via changing molar ratio of monomer repeating unit to achieve the highest protonation. The polymer network having the most suitable stoichiometric ratio for substantial proton conductivity was prepared and characterized by FT-IR spectroscopy and proton conductivity measurements. The network was used for immobilization of invertase and some important kinetic parameters such as the maximum reaction rate (V_max) and Michaelis-Menten constant (K_m) were investigated for the immobilized invertase. Additionally, optimum temperature and pH were determined to acquire the best conditions for the highest enzyme activity. Operational stability of the entrapped enzyme was also examined. The results reveal that the most stable and highly proton conducting polymer network may play a pioneer role in the biosensors applications as given by FT-IR, elemental analysis, impedance spectroscopy and storage stability experiments.     

Novel conducting polymer electrolyte biosensor based on poly(1-vinyl imidazole) and poly(acrylic acid) networks

Biosensor construction and characterization studies of poly(acrylic acid) (PAA) and poly(1-vinyl imidazole) (PVI) complex systems have been carried out. The biosensors were prepared by mixing PAA with PVI at several stoichiometric ratios, x (molar ratio of the monomer repeat units). The enzyme, invertase, was entrapped in the PAA/PVA interpenetrating polymer networks during complexation. Modifications were made on the PAA/PVI conducting polymer electrolyte matrixes to improve the stability and performance of the polymer electrolyte-based enzyme biosensor. The maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) were investigated for the immobilized invertase. The temperature and pH optimization, operational stability, and shelf life of the polymer electrolyte biosensor were also examined.     

Acrylamide grafted poly(ethylene terephthalate) fibers activated by glutaraldehyde as support for urease

Urease was covalently immobilized on acrylamide-grafted poly (ethylene terephthalate) fibers after glutaraldehyde activation. Ureasecontaining fibers showed a very high operational stability and reusability, with about 85% of the initial activity after 90 d. The thermostability of the bound urease was positively influenced, and a slight change in optimum temperature was observed after immobilization, when compared with the free enzyme. The pH optimum of both types of urease was found to be the same, but immobilized urease showed an increased stability in a broader range of pH. The kinetic studies exhibited a slightly higherK _m value for the bound enzyme, with a value of 4.50 mmol dm^-3, when compared with the free enzyme (2.82 mmol dm^-3), which demonstrated that the immobilization procedure did not cause an unfavorable conformation for the substrate-product formation and a hindered diffusion. The graft yield was also found effective on maximum activity of immobilized urease. Twenty-five percent of the acrylamide-grafted fibers exhibited the highest enzymatic activity together with the highest water uptake. Higher graft yields were not suitable for the immobilization of the enzyme molecules as a result of crosslinks formed between the poly(acrylamide) chains and glutaraldehyde.     

Synthesis and characterization of poly(acrylic acid‐co‐acrylamide)/hydrotalcite nanocomposite hydrogels for carbonic anhydrase immobilization

To combine the advantages of a biopolymer with hydrotalcite in an enzyme immobilization system, the intercalation polymerization was used to prepare poly(acrylic acid‐co‐acrylamide)/hydrotalcite (PAA‐AAm/HT) nanocomposite hydrogels using sodium methyl allyl sulfonate as intercalation agent. Transmission electron microscopy, X‐ray diffraction, and Fourier transform infrared spectroscopy results revealed that sodium methyl allyl sulfonate chains entered into the interlayer of HT, the interaction between them has taken place, and HT was dramatically exfoliated into nanoscale and homogeneously dispersed in the PAA‐AAm matrix. Transmission electron microscopy and cryo scanning electron microscope results showed that dried hydrogels were regular spherical particles, and swollen hydrogels revealed homogeneous porous network structures. Then, PAA‐AAm/HT nanocomposite hydrogels were used to immobilize carbonic anhydrase (CA), and the CO₂ hydration activities of free enzyme and immobilized enzyme were evaluated. Results showed that immobilized CA retained the majority of the enzyme activity. The reason may be the formation of a microenvironment almost all of which is composed of free water inside the porous network structures. Therefore, the immobilized CA is of great potential in the removal of trace CO₂ from the closed spaces. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 3232–3240, 2009     

Studies on preparation of metalloporphyrin‐functionalized PVI and PVI/SiO2 via axial coordination reaction

Cobalt tetraphenylporphyrin (CoTPP) was bond on the side chains of poly(N‐vinylimidazole) (PVI) and immobilized on PVI/SiO₂ via axial coordination reactions, respectively, leading to the soluble metalloporphyrin‐functionalized PVI (denoted as CoTPP–PVI) and the particles CoTPP–PVI/SiO₂. The above two target products were characterized using spectroscopy methods such as infrared spectrum, electronic absorption spectrum, and fluorescence emission spectrum. And the effects of various factors on the bonding reaction and immobilization reaction of CoTPP were studied in detail. The experimental results show that CoTPP–PVI and CoTPP–PVI/SiO₂ can be prepared favorably through axial coordination reaction of CoTPP with the side imidazole groups of PVI and PVI/SiO₂. In addition, the soluble CoTPP–PVI has all the spectral characteristics of CoTPP. As compared to CoTPP, the adsorption spectra of CoTPP–PVI exhibit red shift obviously. In the immobilization process of CoTPP on PVI/SiO₂, the higher the concentrations of CoTPP and the reaction temperature, the greater the immobilization amount of CoTPP is. Copyright © 2009 John Wiley & Sons, Ltd.     

Nitroxide spin probe study of probe size,hydrogen bonding and polymer matrix rigidity effects on poly(acrylic acid)/poly(ethylene oxide) complexes

An electron spin resonance (ESR) spin probe study was performed on 1 : 1 by weight poly(acrylic acid) (PAA)/poly(ethylene oxide) (PEO) complex over the 100–450 K temperature range with a series of tetramethylpiperidyloxy‐based spin probes. Measurements of the parameters T_5mT, Ta and Td demonstrated the effects of probe size and the strength of hydrogen bonding. The probes in the series Tempone, Tempo, Tempol and Tamine (respectively 4‐oxo‐, unsubstituted, 4‐hydroxy‐ and 4‐amino‐2,2,6,6,‐tetramethylpiperidine ‐1‐oxyl) displayed noticeable increases in the hydrogen‐bonding effect, as indicated by Ta and Td. These increases correlated with increasing hydrogen bond acceptor strength. On the other hand, as the probe size became larger, T_5mT gradually increased due to the free volume decrease. These effects were analyzed using the established theoretical relationship of T_5mT to probe volume expressed by f. Meanwhile, in order to investigate the effect of polymer matrix rigidity, a similar study was performed with a nitroxide spin probe, 2,2,6,6‐tetramethyl‐1‐piperidine‐1‐oxyl (Tempo), on PAA/PEO complexes of different weight compositions. The quantitative fast motion fraction in the composite ESR spectrum was calculated. The influence of changes in the composition of PAA on the molecular mobility was characterized by changes of the spectral parameters and τ_c. The molecular mobility was shown to diminish with increasing content of PAA in PAA/PEO blends duo to the restriction of the polymer matrix rigidity increase. Copyright © 2003 John Wiley & Sons, Ltd.     

Thermal behaviour of poly(allyl azide)

The paper describes the synthesis of low molecular mass poly(allyl chloride) (PAC) (M _n= 856-3834 g mol^-1) using Lewis acid (ALCL₃, FeCL₃, TiCL₄) and al powder. Branching in PAC was indicated on the basis of elemental analysis and ¹H-NMR spectroscopy. azidation of pac could be carried out at 100°C by using NaN₃ and DMSO as solvent. Curing of poly(allyl azide) (PAA) by cyclic dipolar addition reaction with EGDMA (ethylene glycol dimethacrylate, 5-45 phr) was investigated by differential scanning calorimetry and structure of cured polymer was confirmed by FTIR. A two-step mass loss was exhibited by uncured and cured PAA in nitrogen atmosphere. A mass loss of 20-28% (155-274°C) and 50-61% (330-550°C) was observed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.     

Poly(pyrrole) versus poly(3,4-ethylenedioxythiophene): amperometric cholesterol biosensor matrices

Two conducting polymers, poly(pyrrole) (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT) were used as immobilization matrices for cholesterol oxidase (ChOx). The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as K _m and I _max, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. K _m values are found as 7.9 and 1.3 mM for PPy and PEDOT enzyme electrodes, respectively; it can be interpreted that ChOx immobilized in PEDOT shows higher affinity towards the substrate.     

Miscibility and interactions in blends of two proton‐donating polymers: Poly(acrylic acid)/poly(p‐vinylphenol) blends

Blends of poly(acrylic acid) (PAA) and poly(p‐vinylphenol) (PVPh) were prepared from N,N‐dimethylformamide (DMF) and ethanol solutions. The DMF‐cast blends exhibited single T_g's, as shown by modulated differential scanning calorimetry, whereas the ethanol‐cast blends had double T_g's. Fourier transform infrared spectroscopy showed that there was a specific interaction between PAA and PVPh in the DMF‐cast blends. The single‐T_g blends cast from DMF showed single‐exponential decay behavior for the proton spin–lattice relaxation in both the laboratory frame and the rotating frame, indicating that the two polymers mixed intimately on a scale of 2–3 nm. © 2003 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 41: 789–796, 2003     

About the compatibility of polymer components in polymer complexes based on poly(acrylamide) and poly(vinyl alcohol)

The issue of applying the usual concepts of polymer compatibility to nonstoichiometric PVA/PAA mixtures of chemically complementary poly(vinyl alcohol) and poly(acrylamide), which form in water solution InterPC (intermolecular polymer complex) stabilyzed by H‐bonds, and PAA to PVA graft copolymers (PVA‐PAA_N) with different grafted chains number N, that are IntraPC (intramolecular polymer complexes) is discussed. PVA and PAA are compatible on molecular level. At the same time PVA/PAA mixture (50/50 W/W) is characterized by heterogeneous structure consists of InterPC with ϕ_char=9g_PVA/g_PAA and the excess of unconnected PAA. In the case of IntraPC, yet, only PVA‐PAA_N, where N=25, is characterized by a single glass transition temperature (Tg). At larger values of N separate PAA domains form giving rise to the corresponding Tg. These results are discussed in view of IntraPC structure peculiarities as a function of N investigated by IR spectroscopy.     

Osmotic deswelling of weakly charged poly(acrylic acid) solutions and gels

The osmotic pressure of weakly charged aqueous poly(acrylic acid) (PAA) solutions and the swelling pressure PAA gels were studied by osmotic deswelling at different degrees of ionization (α). In solution, the osmotic pressure was found to scale linearly with concentration, whereas the scaling power of the swelling pressure of gels was higher (1.66). The effect of the ionization degree on the osmotic coefficient in PAA solutions was in agreement with the theory of Borue and Erukhimovich [Macromolecules, 21 , 3240 (1988)]. Ionization increases the swelling capacity of the PAA gels until a plateau is reached at about 35% neutralization. The concentration at equilibrium swelling scales as C_e ～ α^?0.6. The contribution of the network to the gel swelling pressure is evaluated by subtracting the osmotic pressure of the polymer solution at the same concentration and degree of ionization. In swollen gels the extended network opposes swelling. As the gel is osmotically deswelled, a state of zero network pressure exists at a certain concentration, below which the network elasticity favors swelling. The crossover concentration shifts to lower values as the degrees of ionization increases. © 1995 John Wiley & Sons, Inc.     

The aggregation of nonionic surfactants in the presence of poly(methacrylic acid)

The solution properties of homogeneous hexaethylene and octaethylene glycol mono(n-dodecyl) ethers, C₁₂E₆ and C₁₂E₈, respectively, and octaethylene glycol mono(n-decyl) ether, C₁₀E₈, with poly(methacrylic acid) (PMA) were investigated by dye solubilization, surface tension, fluorescence, viscosity, and pH measurements. The data were discussed regarding non-cooperative and cooperative binding of surfactant to polymer. Whereas in the interaction with poly(acrylic acid) (PAA), the critical aggregation concentrations (cac or T ₁) of these surfactants were lower than the respective critical micelle concentration (cmc), in that with the more hydrophobic PMA, T ₁’s of C₁₂E₆ and C₁₂E₈ were higher than the respective cmc, but that of C₁₀E₈ was lower than its cmc. These may be ascribed to the hydrophobic microdomains (HMD) of the PMA coil in water, probably in its inside. It is considered that some surfactants are bound first to the HMD non-cooperatively and then they are abruptly bound cooperatively at T ₁. This raises T ₁ higher than cmc when the cmc is low, and the amount bound by the HMD is relatively large and vice versa. T ₁ of C₁₂E₆ or C₁₂E₈ is the former case, and that of C₁₀E₈ is the latter. Thus, different from PAA, T ₁ for PMA + nonionic surfactant system consists of the amount of non-cooperative binding and the cac of the cooperative binding in equilibrium. Therefore, this T ₁ has a different meaning from that for PAA and should be called apparent T ₁. As the binding to the HMD is dependent on PMA concentration and cac is not, which is like in the PAA system, separation of apparent T ₁ from the HMD binding was achieved by extrapolating T ₁’s to zero PMA concentration (denoted intrinsic T ₁). This value for C₁₂E₈ was found to be lower than the respective cmc and also lower than the respective T ₁ for PAA. With increase in surfactant concentration, the pH of PMA solution rose and demonstrated a peak. This pH rise and fall may be induced by loosening of the HMD coil due to binding increase and by rearrangement of PMA + surfactant complex in high surfactant concentrations region. By raising the initial pH, the HMD were loosened; consequently, T ₁ rose a little, and at higher pH, no surfactant binding took place.     

Synthesis and phase behavior of aqueous poly(N-isopropylacrylamide-co-acrylamide), poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) and poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate)

Poly(N-isopropylacrylamide) (PNIPAM) and random copolymers of Poly(N-isopropylacrylamide-co-2-hydroxyethyl methacrylate) (PNIPAM-HEMA), poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAm), and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (PNIPAM-DMAA) with various volume fractions γ of NIPAM were synthesized by radical polymerization. The phase behavior of the polymers in water was investigated by means of optical transmittance and dynamic light scattering. With decreasing γ, the cloud point temperature T _cp for PNIPAM-HEMA decreased whereas the T _cp for both PNIPAM-AAm and PNIPAM-DMAA increased. Increase of hydrodynamic radius around T _cp, which resulted from the aggregation of the globules of each polymer, was observed from dynamic light scattering. The relationships between the reciprocal of T _cp of the polymer solutions and 1-γ were linear for the three copolymers in the experimental range of 0.65<γ<1. The results are discussed from the aspect of the interaction parameters of copolymer solutions.     

Melting behavior,miscibility, and hydrogen-bonded interactions of poly(ε-caprolactone)/poly(4-hydroxystyrene-co-methoxystyrene) blends

The miscibility of poly(4-hydroxystyrene-co-methoxystyrene) (HSMS) and poly(ε-caprolactone) (PCL) was investigated by differential scanning calorimetry and Fourier transform infrared spectroscopy (FTIR). HSMS/PCL blends were found to be miscible in the whole composition range by detecting only a glass transition temperature (T_g), for each composition, which could be closely described by the Fox rule. The crystallinity of PCL in the blends was dependent on the T_g of the amorphous phase. The greater the HSMS content in the blends, the lower the crystallinity. The polymer–polymer interaction parameter, χ₃₂, was calculated from melting point depression of PCL using the Nishi-Wang equation. The negative value of χ₃₂ obtained for HSMS/PCL blends has been compared with the value of χ₃₂ for poly(4-hydroxystyrene) (P4HS)/PCL blends. The specific nature, quantitative analysis, and average strength of the intermolecular interactions in HSMS/PCL and P4HS/PCL blends have been determined at room temperature and in the molten state by means of Fourier transform infrared spectroscopy (FTIR) measurements. The FTIR results have been in good correlation with the thermal behavior of the blends. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36 : 95–104, 1998     

Synthesis of isotactic,syndiotactic, and heterotactic poly(tributyltin methacrylate) and poly(tributyltin methacrylate-co-styrene)

Pure isotactic and enriched syndiotactic poly(tributyltin methacrylate) were synthesized by the reaction of the respective poly(methacrylic acid) with tributyltin oxide. The heterotactic polymer was prepared in a similar manner and from free radical initiated (AIBN or BPO) polymerization of tributyltin methacrylate. In each case, polymer configuration was confirmed by ¹H-NMR of the hydrolyzed/esterfied product. The relatively large ¹¹⁹Sn-NMR linewidth of the isotactic tributyltin containing polymer suggests an intra-molecular exchange of the pendant tin groups. T_g, T_d, and M _v data are also reported. Poly(tributyltinmethacrylate-co-styrene) was prepared by free radical polymerization and reactivity ratios [r(styrene) = 0.51, r(TBTM) = 0.49] and Q-e values for TBTM (0.78, 0.38) were determined.     

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I_max and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I_max for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.     

Characterization of poly(carboxylic acid)/poly(ethylene oxide) blends formed through hydrogen bonding by spectroscopic and calorimetric analyses

The solid state of the complex between poly(acrylic acid) (PAA) and poly(ethylene oxide) (PEO), and that between poly(methacrylic acid) (PMAA) and PEO formed via hydrogen-bonding was studied by differential-scanning calorimetric (DSC) and by Fourier-transform infrared (FT–IR) spectroscopic measurements. Melting temperature T_m and the degree of the crystallinity X_c of PEO in the systems PAA (or PMAA)/PEO blends obtained from aqueous or dimethyl sulfoxide (DMSO) medium were measured in various unit mol % of PEO ([PEO]100/{[PAA(or PMAA)] + [PEO]}) where [ ] is the unit mole concentration. It was found that 50 unit mol % of PEO is a critical composition, which gives new evidence for the 1 : 1 complex formation between PAA (or PMAA) and PEO. From the FT–IR spectroscopic analysis in conjunction with DSC measurements we also found that the effects of solvent and of hydrophobic interaction (due to the α-methyl group of PMAA) are the important factors controlling the complexation in the solution and solid systems. These factors also affect the crystallization behavior and the microstructure of the PAA (or PMAA)/PEO blend in solid state.     

Thermal degradation of [poly(N-vinylimidazole)-polyacrylic acid] interpolymer complexes

Interpolymer complexes of a slightly basic polymer, poly(N-vinylimidazole) (PVIm) with a strongly acidic polymer, poly(acrylic acid) (PAA) have been prepared by mixing aqueous solutions of the respective components. Spectroscopy and thermal methods were used to reveal interaction between VIm and AA moieties. FT-IR analysis showed that the nitrogen atoms at 3rd position of imidazole ring are involved in strong H-bonding with acid groups of PAA leading to a uniform and fully miscible complex structure. As the quantity of PAA increases the thermal stability of complex increases based on TG results. In the DSC analyses, the single T_g for all IPC samples showed that IPCs have good or definite miscibility over the whole range of composition as a result of H-bond formation between acrylic acid and imidazole units.     

Synthesis,characterization, and swelling behaviors of chitosan‐g‐poly(acrylic acid)/poly(vinyl alcohol) semi‐IPN superabsorbent hydrogels

A series of granulated semi‐interpenetrating polymer network (semi‐IPN) superabsorbent hydrogels composed of chitosan‐g‐poly(acrylic acid) (CTS‐g‐PAA) and poly(vinyl alcohol) (PVA) were prepared by solution polymerization using ammonium persulfate (APS) as an initiator and N,N′‐methylenebisacrylamide (MBA) as a crosslinker. The effects of reaction conditions such as the concentration of MBA, the weight ratio of AA to CTS, and the content of PVA on water absorbency were investigated. Infrared (IR) spectra and differential scanning calorimetry (DSC) analyses confirmed that AA had been grafted onto CTS backbone, and PVA semi‐interpenetrating into CTS‐g‐PAA networks. SEM analyses indicated that CTS‐g‐PAA/PVA has improved porous surface and PVA was uniformly dispersed in CTS‐g‐PAA network. The semi‐IPN hydrogel containing 10 wt% PVA shows the highest water absorbency of 353 and 53 g g^?1 in distilled water and 0.9 wt% NaCl solution, respectively. Swelling behaviors revealed that the introduction of PVA could improve the swelling rate and enhance the pH stability of the superabsorbent hydrogel. Copyright © 2009 John Wiley & Sons, Ltd.