Novel photochemical surface functionalization of polysulfone ultrafiltration membranes for covalent immobilization of biomolecules

The major objective of the work was to develop a heterogeneous modification method for attachment of reactive groups, suitable for covalent immobilization of active biomolecules on the surface of polysulfone ultrafilters without loss of membrane selectivity. For applying a polymer specific activation chemistry, the materials of commercial “polysulfone” UF membranes were identified using elemental analysis along with ¹H NMR, FTIR-ATR and UV spectroscopy. Heterogeneous photoinitiated graft polymerization was realized using acrylic acid (AA) as model monomer and as carrier of reactive groups. Polymer structure (polysulfone, PSf, or polyethersulfone, PESf), coating with photoinitiator (benzophenone, BP, or benzoylbenzoic acid, BPC) and UV excitation energy (λ_exc220> 300 or 350 nm) were the major parameters. Grafted polyAA (g-PAA) could be obtained under almost all conditions but with largely varying yields (DG). However, only with λ_exc350 nm, polymer and pore degradation could be excluded. A new selective initiation of graft polymerization onto PSf, H-abstraction by photoexcited BP derivatives from the methyl side groups, thus avoiding polymer chain scission, was proved indirectly. Modified structures were characterized spectroscopically, including visualization with SFM of laterally patterned surfaces generated by UV irradiation through a mask. UF tests of PSf-g-PAA and PESf-g-PAA UF membranes (DG 100…150 μg/cm²), prepared under “mildly degrading” conditions (λ_exc300 nm), indicated only slight permeability and selectivity changes compared with unmodified samples. Selective PSf functionalization (BPC coating, λ_exc350 nm; DG 5 μg/cm²) caused flux reductions and dextran selectivity increases by factors of 1.3. Covalent immobilization onto g-PAA-functionalized and carbodiimide-activated PSf or PESf membrane surfaces was studied with a protein (BSA), an enzyme (invertase, INV), an antibody-enzyme (IgG-POD) conjugate, and a peptide (“PC1”) as specific antigen of a monoclonal antibody. High binding capacities, up to 40 fold compared with a flat unmodified surface, were detected either directly (BSA) or indirectly via specific activity/binding assays (INV, IgG-POD, “PC1”). This indicated an increased outer membrane surface area due to multifunctional reactive and hydrophilic grafted polymer chains.     

New insights from X-ray photoelectron spectroscopy into the chemistry of covalent enzyme immobilization, with glutamate dehydrogenase (GDH) on silicon dioxide as an example

A three-step process for immobilization of glutamate dehydrogenase (GDH) on the surface of silicon dioxide has been studied by X-ray photoelectron spectroscopy (XPS). The enzyme layer was deposited on the silicon dioxide surface after first exposing the surface to 3-aminopropyltriethoxysilane (3-APTS) and reacting the silylated surface with glutaraldehyde (GA). Fine XPS analysis, performed after each step of the chemical procedure, revealed unknown details of the step-by-step construction of the enzyme layer under different experimental conditions.     

Preparation and characterization of stable chitosan nanofibrous membrane for lipase immobilization

Nanofibrous membrane with a fiber diameter of 80-150 nm was fabricated from mixed chitosan/poly(vinyl alcohol) (PVA) solution by an electrospinning process. Field emission scanning electron microscope and transmission electron microscope were used to characterize the morphology of the nanofibrous membrane. It was found that chitosan nanofibrous membrane with stabilized morphology could be prepared through removing most of PVA from the nascent one with 0.5 M NaOH aqueous solution. This treatment also resulted in an obvious decrease in fiber diameter. The stabilized chitosan nanofibrous membrane was explored as support for enzyme immobilization due to the characteristics of excellent biocompatibility, high surface/volume ratio, and large porosity. Lipase from Candida rugosa was immobilized on the nanofibrous membrane using glutaraldehyde (GA) as coupling reagent. The properties of the immobilized lipase were assayed and compared with the free one. Results showed that, the observed lipase loading on this nanofibrous membrane was up to 63.6 mg/g and the activity retention of the immobilized lipase was 49.8% under the optimum condition. The pH and thermal stabilities of lipase were improved after it was immobilized on the chitosan nanofibrous membrane. In addition, the experimental results of reusability and storage stability indicated that the residual activities of the immobilized lipase were 46% after 10 cycles and 56.2% after 30 days, which were obviously higher than that of the free one.     

Enzyme immobilization on a pH-responsive porous polymer membrane for enzymatic kinetics study

Immobilization of enzymes onto carriers is a rapidly growing research area aimed at increasing the stability, reusability and enzymolysis efficiency of free enzymes. In this work, the role of phase-separation and a pH-responsive “hairy” brush, which greatly affected the topography of porous polymer membrane enzyme reactors (PMER), was explored. The porous polymer membrane was fabricated by phase-separation of poly(styrene-co-maleic anhydride-acrylic acid) and poly(styrene-ethylene glycol). Notably, the topography and pores size of the PMER could be controlled by phase-separation and a pH-responsive “hairy” brush. For evaluating the enzymolysis efficiency of d-amino acid oxidase (DAAO) immobilized carrier (DAAO@PMER), a chiral ligand exchange capillary electrophoresis method was developed with d-methionine as the substrate. The DAAO@PMER showed good reusability and stability after five continuous runs. Notably, comparing with free DAAO in solution, the DAAO@PMER exhibited a 17.7-folds increase in catalytic velocity, which was attributed to its tailorable topography and pH-responsive property. The poly(acrylic acid) moiety of poly(styrene-co-maleic anhydride-acrylic acid) as the pH-responsive “hairy” brush generated topography changing domains upon adjusting the buffer pH, which enable the enzymolysis efficiency of DAAO@PMER to be tuned based upon the well-defined architectures of the PMER. This approach demonstrated that the topographical changes formed by phase-separation and the pH-responsive “hairy” brush indeed made the proposed porous polymer membrane as suitable supports for enzyme immobilization and fitting for enzymolysis applications, achieving high catalytic performance.     

Functionalization of cellulose dialysis membranes for chiral separation using beta-cyclodextrin immobilization

A membrane-based chiral separation system for the separation of racemic tryptophan solutions is developed by the covalently binding beta-cyclodextrin onto the surface of commercial cellulose membranes. The immobilization process is monitored by XPS. AFM demonstrates the evolutionary transition of membrane surface morphology before and after the CD immobilization. Due to their different complexation with immobilized CD, dialysis transport experiments show d-tryptophan preferential permeability through the immobilized CD membranes, and the enantioselectivity is 1.10. A model based on the existence of a thin chiral solution layer of amino acid at the interface between the feed solution and the membrane has been proposed. This chiral separation model has been verified using the chiral separation results of racemic amino acids and binding constants of amino acids with CD. The effect of membrane's pore size on enantioselectivity has also been investigated. The immobilized CD membrane, having MWCO 1000, exhibits the highest enantioselectivity to the racemic tryptophan solution.     

Surface functionalization of oxidized multi-walled carbon nanotubes: Candida rugosa lipase immobilization

This work examines two approaches for immobilization of lipase from Candida rugosa on oxidized multi-walled carbon nanotubes (o-MWCNTs). One method included the presence of activating agents to promote covalent bonding and the other the adsorption on o-MWCNTs to elucidate if non-specific bonding on the o-MWCNTs surface exists. The influence of the immobilization time and initial enzyme concentration on protein loading and the expressed lypolitic activity of the immobilized preparation were investigated. The results showed that the enzyme adsorbs on o-MWCNTs in a maximal amount of 37 μg mg⁻¹ CNTs, while the attached amount was more than 2-times higher under covalent promoting conditions (80 μg mg⁻¹ CNTs). Furthermore, similar trends were observed for the lypolitic activity, whereby preparations obtained under covalent promoting conditions had almost 3-times higher activity (560 IU g⁻¹ of immobilized enzyme). In addition, immobilization of the enzyme was confirmed by Fourier transformation infrared spectroscopy and thermogravimetric analysis.     

Photochemical activation of a polycarbonate surface for covalent immobilization of a protein ligand

Polycarbonate—a thermostable polymer is activated by a simple and rapid method using a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) for covalent immobilization of a biomolecule. Horseradish peroxidase (HRP) is used as a model enzyme to check the efficacy of the activated surface. HRP is immobilized on the activated polycarbonate surface without addition of any reagent or catalyst and is found to give 2-2.5-fold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. Photochemical attachment of FNAB to the PC surface is confirmed by X-ray photoelectron spectroscopy (XPS), which shows the presence of nitrogen and fluorine in the ratio of 2:1 in the activated polycarbonate. Disappearance of fluorine peak in the XP spectra of PC bound enzyme further confirms the covalent binding of HRP, through displacement of fluorine moiety of the activated PC by the amino group of the protein. Optimized concentration of the photolinker is found as 6 μmol of FNAB per well and time of photo irradiation is 8 min for activation of a PCR polycarbonate plate. PC bound HRP has shown enhanced thermal and storage stability. Kinetic studies of the immobilized HRP shows improved catalytic activity. The potential application of activated polycarbonate surface includes immobilization of biomolecules for biosensors, immunoassays, and protein and DNA micro-arrays. Due to the stability of the polycarbonate at high temperature, the activated polycarbonate has an advantage for immobilization of thermostable biomolecule such as thermostable enzyme for reaction at elevated temperature.     

The effect of covalent surface immobilization on the bactericidal efficacy of a quaternary ammonium compound

This study investigates the effect of surface immobilization on the bactericidal function of a quaternary ammonium compound. Quaternary ammonium silane (QAS) coated planar surfaces did not produce any measurable mortality of Staphylococcus aureus, while 1 µm QAS‐coated microparticles did produce S. aureus mortality. The experiments using QAS‐coated microparticles indicate that the ability of QAS molecules to disrupt the cell wall is not hindered by covalent immobilization of QAS to a surface. These results provide evidence that S. aureus cells on a QAS‐coated planar surface are not exposed to a sufficient number of QAS molecules to produce significant mortality. This result has important implications for the development of self‐decontaminating coatings. Covalent immobilization is used to prevent leaching of the bactericidal compound. However, covalent immobilization may result in a significant tradeoff in bactericidal performance. Published in 2007 by John Wiley & Sons, Ltd.     

Electrochemical sensing platform based on covalent immobilization of thionine onto gold electrode surface via diazotization-coupling reaction

A novel electrochemical sensing platform by modification of electroactive thionine (Th) onto gold electrode surface was constructed, which was realized by diazotization of 4-aminothiophenol (ATP) self-assembled monolayer, followed by coupling of Th with the diazonium group to form a covalent diazo bond. A pair of well-defined redox peaks of Th was observed in the cyclic voltammetric measurement. The resulting diazo-ATP monolayer displayed superior electrical conductivity, which contributed to the sensitive detection of hydrogen peroxide (H₂O₂). The immobilized Th also showed a remarkable stability, which may benefit from the π-π stacking force and the covalent diazo bond between diazo-ATP and Th molecules. Under the optimized experimental conditions, the current fabricated non-enzyme and reagentless sensor could show a rapid response to H₂O₂ within 3 s and a linear calibration plot ranged from 1.0 × 10⁻⁶ to 6.38 × 10⁻³ M with a detection limit of 6.7 × 10⁻⁷ M. The current fabrication strategy of electroactive interface is expected to be used as a versatile route for the immobilization of more electroactive molecules and offer more opportunities for the applications in electrochemical sensor, biosensor, electrocatalysis, etc.     

A novel fluorescence sensor based on covalent immobilization of 3-amino-9-ethylcarbazole by using silver nanoparticles as bridges and carriers

A novel technique of covalent immobilization of indicator dyes in the preparation of fluorescence sensors is developed. Silver nanoparticles are used as bridges and carriers for anchoring indicator dyes. 3-amino-9-ethylcarbazole (AEC) was employed as an example of indicator dyes with terminal amino groups and covalently immobilized onto the outmost surface of a quartz glass slide. First, the glass slide was functionalized by (3-mercaptopropyl) trimethoxysilane (MPS) to form a thiol-terminated self-assembled monolayer, where silver nanoparticles were strongly bound to the surface through covalent bonding. Then, 16-mercaptohexadecanoic acid (MHDA) was self-assembled to bring carboxylic groups onto the surface of silver nanoparticles. A further activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) converted the carboxylic groups into succinimide esters. Finally, the active succinimide esters on the surface of silver nanoparticles were reacted with AEC. Thus, AEC was covalently bound to the glass slide and an AEC-immobilized sensor was obtained. The sensor exhibited very satisfactory reproducibility and reversibility, rapid response and no dye-leaching. Rutin can quench the fluorescence intensity of the sensor and be measured by using the sensor. The linear response of the sensor to rutin covers the range from 2.0 × 10⁻⁶ to 1.5 × 10⁻⁴ mol L⁻¹ with a detection limit of 8.0 × 10⁻⁷ mol L⁻¹. The proposed technique may be feasible to the covalent immobilization of other dyes with primary amino groups.     

A new method for reversible immobilization of thiol biomolecules based on solid-phase bound thiolsulfonate groups

A new method for the reversible immobilization of thiol bimolecules, e.g., thiolpeptides and thiolproteins, to beaded agarose and other solid phases is reported. The method consists of an activation and a coupling step. The activation is based on oxidation of disulfides (or thiol groups via disulfides) present in a solid phase by hydrogen peroxide at moderately acidic pH. This oxidation leads to disulfide oxides (thiolsulfinate groups of which the majority are further oxidized to thiolsulfonate). The thiolsulfonate groups react easily with thiol compounds, which become immobilized via disulfide bonds. The pH range for thiol coupling is wide (pH 5-8), but for most thiols the reaction seems to proceed faster at pH>7. The stability of the reactive group to hydrolysis, especially at neutral and weakly acidic pH, is very high. The activated gel, therefore, can be stored as a suspension at pH 5 for extended periods. The method has been used to reversibly immobilize glutathione, β-galactosidase, alcohol dehydrogenase, urease, and papain, all with exposed thiol groups as well as thiolated bovine serum albumin and sweet-potato β-amylase. Depending on the thiol content of starting thiol-agarose, thiol-sulfonate-agarose derivatives with different binding capacities can be obtained. Thus, up to 5.0 mg (16 μmol) glutathione and 15 mg thiol-protein/mL gel derivative have been immobilized.     

Development of hydrogen peroxide biosensor based on in situ covalent immobilization of horseradish peroxidase by one-pot polysaccharide-incorporated sol-gel process

A simple and reliable one-pot approach was established for the development of a novel hydrogen peroxide (H₂O₂) biosensor based on in situ covalent immobilization of horseradish peroxidase (HRP) into biocompatible material through polysaccharide-incorporated sol-gel process. Siloxane with epoxide ring and trimethoxy anchor groups was applied as the bifunctional cross-linker and the inorganic resource for organic-inorganic hybridization. The reactivity between amine groups and epoxy groups allowed the covalent incorporation of HRP and the functional biopolymer, chitosan (CS) into the inorganic polysiloxane network. Some experimental variables, such as mass ratio of siloxane to CS, pH of measuring solution and applied potential for detection were optimized. HRP covalently immobilized in the hybrid matrix possessed high electrocatalytic activity to H₂O₂ and provided a fast amperometric response. The linear response of the as-prepared biosensor for the determination of H₂O₂ ranged from 2.0 × 10⁻⁷ to 4.6 × 10⁻⁵ mol l⁻¹ with a detection limit of 8.1 × 10⁻⁸ mol l⁻¹. The apparent Michaelis-Menten constant was determined to be 45.18 μmol l⁻¹. Performance of the biosensor was also evaluated with respect to possible interferences. The fabricated biosensor exhibited high reproducibility and storage stability. The ease of the one-pot covalent immobilization and the biocompatible hybrid matrix serve as a versatile platform for enzyme immobilization and biosensor fabricating.     

A new simple preparation of platinum-nickel alloy nanoparticles and their characterization as an electrocatalyst for methanol oxidation

Pt-Ni alloy nanoparticles were produced by casting 2 or 10 mM H₂PtCl₆ solutions on a Ni column. The apparent particle size for the resultant Pt-Ni alloys increased with the concentration of the H₂PtCl₆ solution, while the content of Pt in the alloy decreased. The potential sweeps of 5 cycles in an H₂SO₄ aqueous solution for Pt-Ni (2 mM)/Ni and Pt-Ni (10 mM)/Ni electrodes led to electrochemical behavior similar to a polycrystalline Pt electrode, suggesting the formation of a few thin Pt layers on each Pt-Ni alloy surface. In electrochemical measurements, both Pt-Ni/Ni electrodes showed more negative onset potential of methanol oxidation and slower degradation of oxidation current of methanol than the polycrystalline Pt electrode. X-ray photoelectron spectroscopy of both Pt-Ni/Ni electrodes showed the shift of Pt4f peaks to a higher binding energy, suggesting that the increase in the d vacancy in the balance band 5d orbital of Pt contributed to the improved electrocatalytic activity and durability of the Pt-Ni/Ni electrodes.     

Fluorescence sensor for water in organic solvents prepared from covalent immobilization of 4-morpholinyl-1, 8-naphthalimide

A new fluorescent dye, N-allyl-4-morpholinyl-1,8-naphthalimide (AMN), was synthesized as a fluorescence indicator in the fabrication of a sensor for determining water content in organic solvents. To prevent leakage of the fluorophore, AMN was photo-copolymerized with acrylamide, (2-hydroxyethyl)methacrylate, and triethylene glycol dimethacrylate on a glass surface treated with a silanizing agent. The sensing mechanism is based on the solvatochromic feature of the covalently immobilized AMN. The fluorescence intensity of AMN decreased with increasing water contents when it was excited at 400 nm. In the range of ca. 0.00–4.40% (v/v), the fluorescence intensity of AMN changed as a linear function of water content. The sensor exhibited satisfactory reproducibility, reversibility, and a response time (t ₉₉) of the order of 50 s. The detection limit was solvent-dependent, when acetonitrile was used as the solvent, and the detection limit could be as low as 0.006% (v/v) of water. Additionally, the prepared sensor is pH-insensitive and possesses a relatively long lifetime of at least one month.     

Organic–inorganic hybrid silica as supporting matrices for selective recognition of bovine hemoglobin via covalent immobilization

The synthesis of poly‐aminophenylboronic acid (APBA) imprinted hybrid silica‐based polymers for selective recognition of bovine hemoglobin (BHb) was described, where the mesoporous hybrid silica supporting matrices were prepared by a mild sol–gel process with tetraethoxysilane and 3‐aminopropyltriethoxysilane as two precursors. Covalent immobilization of BHb was adopted in order to create homogeneous recognition sites. After removal of the template, the resulting imprinted polymers showed high binding affinity toward BHb and the imprinting factor (α) reached 2.12. The specificity of the BHb recognition was evaluated with competitive experiments, indicating the imprinted polymers have a higher selectivity for the template BHb. The easy preparation protocol and good protein recognition properties made the approach an attractive solution to depletion of high‐abundance protein from bovine blood.     

Novel cellulose acetate membrane blended with phospholipid polymer for hemocompatible filtration system

To improve the blood compatibility of cellulose acetate (CA) membranes for hemofiltration, a novel CA membrane blended with 2-methacryloyloxyethyl phosphorylcholine (MPC) copolymer was designed for a hemocompatible filtration system. The MPC copolymer (PMB30) was synthesized from MPC and n-butyl methacrylate. The polymer solution for making the membrane was prepared from a solvent mixture composed of N,N-dimethylformamide, acetone, and 2-propanol. The CA and CA/PMB30 blended membranes with an asymmetric and porous structure were prepared by a phase inversion process. The mechanical properties and solute permeability of the CA/PMB30 blended membrane could be controlled by preparation conditions such as the composition of the solvents and the solvent evaporation time. The CA/PMB30 blended membrane showed both good water and solute permeabilities in comparison with the CA membrane. Also, the molecular weight of the solute passed through the membrane was changed by the addition of PMB30, and good permselectivity could be obtained. Moreover, the CA/PMB30 blended membranes had excellent blood compatibility such as protein adsorption resistivity compared to the CA membrane due to location of the MPC units in the PMB30 at the surface.