Behavior of glucose oxidase immobilized in various electropolymerized thin films

Glucose oxidase was immobilized by electropolymerization into films of polyaniline, polyindole, polypyrrole, poly(o-phenylediamine), and polyaniline crosslinked with p-phenylenediamine. The kinetics and the behavior of the entrapped enzyme toward elevated temperature, organic solvent denaturation, and pH were investigated, along with the response of the films to electroactive species such as acetaminophen, ascorbate, cysteine, and uric acid. For most of the films, linearity to glucose extended from 7 to 10 mM. The poly(o-phenylenediamine)/glucose oxidase film gave the best signal/noise ratio and polypyrrole/glucose oxidase film gave the most reproducible current responses. No significant shift of the optimum reaction pH (5.5), except for polypyrrole (5.0), was observed after immobilization of glucose oxidase in the various films. Enzymatic activity decreased rapidly when pH was raised above 7.5. Thermodeactivation studies at 55 degrees , 60 degrees , and 65 degrees C have shown polypyrrole/and poly(o-phenylediamine)/glucose oxidase films to be the most resistant enzymatic films. Poly(o-phenylenediamine) films offered the best protection against glucose oxidase deactivation in hexane, chloroform, ether, THF, and acetonitrile when compared with the other electropolymerized films. All the enzymatic films exhibited permselection toward electroactive species. (c) 1996 John Wiley & Sons, Inc.     

AFM nanometer surface morphological study of in situ electropolymerized neutral red redox mediator oxysilane sol-gel encapsulated glucose oxidase electrochemical biosensors

Four different silica sol-gel films: methyltrimethoxysilane (MTMOS), tetraethoxysilane (TEOS), 3-aminopropyltriethoxysilane (APTOS) and 3-glycidoxypropyl-trimethoxysilane (GOPMOS) assembled onto highly oriented pyrolytic graphite (HOPG) were characterized using atomic force microscopy (AFM), due to their use in the development of glucose biosensors. The chemical structure of the oxysilane precursor and the composition of the sol-gel mixture both influenced the roughness, the size and the distribution of pores in the sol-gel films, which is relevant for enzyme encapsulation. The GOPMOS sol-gel film fulfils all the morphological characteristics required for good encapsulation of the enzyme, due to a smooth topography with very dense and uniform distribution of only small, 50nm diameter, pores at the surface. APTOS and MTMOS sol-gel films developed small pores together with large ones of 300-400nm that allow the leakage of enzymes, while the TEOS film formed a rough and incomplete network on the electrode, less suitable for enzyme immobilisation. GOPMOS sol-gel film with encapsulated glucose oxidase and poly(neutral red) redox mediator, prepared by in situ electropolymerization, were also morphologically characterized by AFM. The AFM results explain the variation of the stability in time, sensitivity and limit of detection obtained with different oxysilane sol-gel encapsulated glucose oxidase biosensors with redox mediator.     

Response of cells on surface-induced nanopatterns: fibroblasts and mesenchymal progenitor cells

Ultrathin films of a poly(styrene)-block-poly(2-vinylpyrindine) diblock copolymer (PS-b-P2VP) and poly(styrene)-block-poly(4-vinylpyrindine) diblock copolymer (PS-b-P4VP) were used to form surface-induced nanopattern (SINPAT) on mica. Surface interaction controlled microphase separation led to the formation of chemically heterogeneous surface nanopatterns on dry ultrathin films. Two distinct nanopatterned surfaces, namely, wormlike and dotlike patterns, were used to investigate the influence of topography in the nanometer range on cell adhesion, proliferation, and migration. Atomic force microscopy was used to confirm that SINPAT was stable under cell culture conditions. Fibroblasts and mesenchymal progenitor cells were cultured on the nanopatterned surfaces. Phase contrast and confocal laser microscopy showed that fibroblasts and mesenchymal progenitor cells preferred the densely spaced wormlike patterns. Atomic force microscopy showed that the cells remodelled the extracellular matrix differently as they migrate over the two distinctly different nanopatterns.     

Cholesterol biosensors prepared by layer-by-layer technique

The analysis of formation, deposition and characterization of cholesterol oxidase (COX) layer-by-layer films were performed. Initially, a layer of polyanion, poly(styrene sulfonate) (PSS) was adsorbed followed by a layer of polycation, poly(ethylene imine) (PEI) on each solid substrate from aqueous solutions. The alternating layers were formed by consecutive adsorption of polycations (PEI) and negatively charged proteins (COX) and cholesterol esterase (CE). A strong interaction between protein and polyelectrolyte improves the stability of the alternating multilayer; however, it can change a native protein conformation and impair the protein activity. The PSS/PEI/COX, PSS/PEI/COX/PEI/CE, PSS/PEI/COX-CE/PEI etc. layered structures were prepared on the surface of a platinum electrode, ITO coated glass plate, quartz crystal microbalance, quartz plates, mica and silicon substrates. Optical and gravimetric measurements based on an ultraviolet–visible absorption spectroscopy and a quartz crystal microbalance revealed that the enzyme multilayers thus prepared consist of molecular layered of the proteins. The surface morphology of such bilayer films was investigated by using atomic force microscopy. The electrochemical redox processes of the enzyme-layered films deposited either on platinum or ITO coated glass plate were investigated. The response current of cholesterol oxidase electrode with concentration of cholesterol was investigated at length.     

The modification of enzyme electrode properties with non-conducting electropolymerised films

Phenol and several phenol derivatives have been electropolymerised on to platinum anodes. The selective properties of the modified electrodes were dependent on the final polymeric structure. Although essentially non-conducting, dual phenolic films could be deposited on electrode surfaces. This was achieved by the initial deposition of a porous polymer formed from, phenol red, followed by the more diffusion limiting film of oxidised phenol. The resulting composite film showed the combined selective characteristics of the two component polymers. Attempts to enhance polymeric film selectivity via the incorporation of charged or neutral surfactants are outlined. The characteristics of glucose oxidase incorporated into (poly)phenol films are discussed.     

Reactive thin polymer films as platforms for the immobilization of biomolecules

Spin-coated thin films of poly(N-hydroxysuccinimidyl methacrylate) (PNHSMA) on oxidized silicon and gold surfaces were investigated as reactive layers for obtaining platforms for biomolecule immobilization with high molecular loading. The surface reactivity of PNHSMA films in coupling reactions with various primary amines, including amine-terminated poly(ethylene glycol) (PEG-NH2) and fluoresceinamine, was determined by Fourier transform infrared (FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and ellipsometry measurements, respectively. The rate constants of PEG-NH2 attachment on the PNHSMA films were found to be significantly increased compared to the coupling on self-assembled monolayers (SAMs) of 11,11'-dithiobis(N-hydroxysuccinimidylundecanoate) (NHS-C10) on gold under the same conditions. More significantly, the PEG loading observed was about 3 times higher for the polymer thin films. These data indicate that the coupling reactions are not limited to the very surface of the polymer films, but proceed into the near-surface regions of the films. PNHSMA films were shown to be stable in contact with aqueous buffer; the swelling analysis, as performed by atomic force microscopy (AFM), indicated a film thickness independent swelling of approximately 2 nm. An increased loading was also observed by surface plasmon resonance for the covalent immobilization of amino-functionalized probe DNA. Hybridization of fluorescently labeled target DNA was successfully detected by fluorescence microscopy and surface plasmon resonance enhanced fluorescence spectroscopy (SPFS), thereby demonstrating that thin films of PNHSMA comprise an attractive and simple platform for the immobilization of biomolecules with high densities.     

Amperometric glucose biosensor based on multilayer films via layer-by-layer self-assembly of multi-wall carbon nanotubes, gold nanoparticles and glucose oxidase on the Pt electrode

A novel amperometric glucose biosensor based on the nine layers of multilayer films composed of multi-wall carbon nanotubes (MWCNTs), gold nanoparticles (GNp) and glucose oxidase (GOD) was developed for the specific detection of glucose. MWCNTs were chemically modified with the H₂SO₄–HNO₃ pretreatment to introduce carboxyl groups which were used to interact with the amino groups of poly(allylamine) (PAA) and cysteamine via 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide cross-linking reaction, respectively. A cleaned Pt electrode was immersed in PAA, MWCNTs, cysteamine and GNp, respectively, followed by the adsorption of GOD, assembling the one layer of multilayer films on the surface of Pt electrode (GOD/GNp/MWCNTs/Pt electrode). Repeating the above process could assemble different layers of multilayer films on the Pt electrode. PBS washing was applied at the end of each assembly deposition for dissociating the weak adsorption. Film assembling and characterization were studied by transmission electron microscopy and quartz crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The marked electrocatalytic activity of Pt electrode based on multilayer films toward H₂O₂ produced during GOD enzymatic reactions with glucose permitted effective low-potential amperometric measurement of glucose. Taking the sensitivity and selectivity into consideration, the applied potential of 0.35 V versus Ag/AgCl was chosen for the oxidation detection of H₂O₂ in this work. Among the resulting glucose biosensors, the biosensor based on nine layers of multilayer films was best. It showed a wide linear range of 0.1–10 mM glucose, with a remarkable sensitivity of 2.527 μA/mM, a detection limit of 6.7 μM estimated at a signal-to-noise ratio of 3 and fast response time (within 7 s). Moreover, it exhibited good reproducibility, long-term stability and the negligible interferences of ascorbic acid, uric acid and acetaminophen. The study can provide a feasible approach on developing new kinds of oxidase-based amperometric biosensors, and can be used as an illustration for constructing various hybrid structures.     

Amperometric glucose biosensor based on layer-by-layer assembly of multilayer films composed of chitosan, gold nanoparticles and glucose oxidase modified Pt electrode

A new strategy for fabricating glucose biosensor was presented by layer-by-layer assembled chitosan (CS)/gold nanoparticles (GNp)/glucose oxidase (GOD) multilayer films modified Pt electrode. First, a cleaned Pt electrode was immersed in poly(allylamine) (PAA), and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/PAA/Pt). Second, the GOD/GNp/PAA/Pt electrode was immersed in CS, and then transferred to GNp, followed by the adsorption of GOD (GOD/GNp/CS/GOD/GNp/PAA/Pt). Third, different layers of multilayer films modified Pt electrodes were assembled by repeating the second process. Film assembling and characterization were studied by quart crystal microbalance, and properties of the resulting glucose biosensors were measured by electrochemical measurements. The results confirmed that the assembling process of multilayer films was simple to operate, the immobilized GOD displayed an excellent catalytic property to glucose, and GNp in the biosensing interface efficiently improved the electron transfer between analyte and electrode surface. The amperometric response of the biosensors uniformly increased from one to six layers of multilayer films, and then reached saturation after the seven layers. Among the resulting biosensors, the biosensor based on the six layers of multilayer films was best. It showed a wide linear range of 0.5-16 mM, with a detection limit of 7.0 microM estimated at a signal-to-noise ratio of 3, fast response time (within 8s). Moreover, it exhibited good reproducibility, long-term stability and interference free. This method can be used for constructing other thin films, which is a universal immobilization method for biosensor fabrication.     

Fluidics-resolved estimation of protein adsorption kinetics in a biomicrofluidic system

Protein adsorption on surfaces is a complex phenomenon that is described by the balance of convective/diffusive transport of the protein species to the surface and its adsorption/desorption at the surface. The extent of binding depends on a variety of factors such as protein/surface interactions, availability of binding sites, localized concentrations of protein near biomaterial surfaces and flow characteristics of the protein in that region. Factors such as time-varying flows, complex device geometries, presence of multiple competitive species, or possible denaturing of proteins when they attach to the surface make it extremely difficult to quantitatively analyze protein interactions with surfaces. Adsorption/desorption rate constants are often inferred using simplistic models which neglect mass transport and have limited use across different microfluidic systems and flow protocols. In this work, we have developed and demonstrated a fluidics-resolved model that evaluates protein adsorption, accounting for both the fluidic transport and the biochemical kinetics in complex biomicrofluidic devices. The model is valid for both flow and static conditions. An automated procedure was also developed to extract the "intrinsic" mass-transport-independent adsorption kinetic rate constants from experimental data using a least squares optimization method. The automated data extraction methodology is applied to two proteins (alkaline phosphatase and glucose oxidase) that have been brought into contact with poly(etheretherketone) and Teflon capillaries. The applicability of the procedure in analyzing flow and adsorption in complex microfluidic structures is also demonstrated.     

Radical graft polymerization of styrene sulfonate on poly(ethylene terephthalate) films for ACL applications: "grafting from" and chemical characterization

The purpose of this study is to develop a reliable method of functionalizing poly(ethylene terephthalate) with bioactive polymers to produce a "biointegrable" artificial anterior cruciate ligament. Radical graft polymerization of the sodium salt of styrene sulfonate (NaSS) onto poly(ethylene terephthalate) (PET) films was performed using the "grafting from" technique. Prior to the grafting, the surfaces of poly(ethylene terephthalate) films were activated by ozonation to generate peroxide and hydroperoxide reactive species on the PET film surfaces. The radical polymerization of NaSS was initiated by thermal decomposition of the hydroperoxides. The grafted PET surfaces were characterized by a toluidin blue colorimetric method, X-ray photoelectron spectroscopy, contact angle measurements, and atomic force microscopy. The influence of ozonation time, monomer concentration, and temperature on NaSS grafting ratios was examined. A total of 30 min of ozonation followed by grafting from a 15% NaSS solution at 70 degrees C for 90 min or more resulted in attachment of poly(NaSS) chains to the PET film surfaces.     

Carbon nanotubes promote neuron differentiation from human embryonic stem cells

Human embryonic stem cells (hESCs) hold great promise for regenerative medicine and transplantation therapy due to their self-renewal and pluripotent properties. We report that 2D thin film scaffolds composed of biocompatible polymer grafted carbon nanotubes (CNTs), can selectively differentiate human embryonic stem cells into neuron cells while maintaining excellent cell viability. According to fluorescence image analysis, neuron differentiation efficiency of poly(acrylic acid) grafted CNT thin films is significant greater than that on poly(acrylic acid) thin films. When compared with the conventional poly-l-ornithine surfaces, a standard substratum commonly used for neuron culture, this new type thin film scaffold shows enhanced neuron differentiation. No noticeable cytotoxic effect difference has been detected between these two surfaces. The surface analysis and cell adhesion study have suggested that CNT-based surfaces can enhance protein adsorption and cell attachment. This finding indicates that CNT-based materials are excellent candidates for hESCs’ neuron differentiation.     

Glucose sensor using a microfabricated electrode and electropolymerized bilayer films

A new type miniaturized glucose sensor with good selectivity and stable current response has been developed. The structure consists of a recessed rectangular microfabricated platinum electrode, inner layer of two electropolymerized nonconducting films, and outer bilayer of poly(tetrafluoroethylene) (Teflon) and polyurethane (PU) films. Glucose oxidase (GOx) is entrapped during the electropolymerization of a poly(m-phenylenediamine) (PMPD) film in an acetate buffer (AB) solution, on which a highly interference-resistive PMPD film is deposited in a phosphate buffered saline (PBS) solution. The second PMPD film causes no significant decrease in accessibility of glucose to GOx. The inner layer maintains less than 1% permeability to acetaminophen for 12 days. The fairly adhesive outer layer allows stable current response. Due to high permeability, the information about enzyme activity can be obtained without serious error in spite of outer layer intervening between enzymes and solution. The apparent Michaelis-Menten constant and the maximum steady-state current density were 24 mM and 80 microA cm(-2), respectively.     

Sequential actions of glucose oxidase and peroxidase in molecular films assembled by layer-by-layer alternate adsorption

Molecular films of protein/polyion layers were assembled by means of alternate adsorption through electrostatic interaction. Glucose oxidase (GOD) and peroxidase (POD) were assembled in combination with sodium poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI), respectively. Enzyme activities of those films on specific substrates (glucose and H(2)O(2)) were examined by coloring reaction of dye DA67. A multienzyme film containing GOD layer and POD layer was prepared by alternate adsorption of POD/PSS followed by PEI/GOD. Sequential redox reaction of glucose/H(2)O(2)/DA67 was demonstrated successfully with this supramolecular system. (c) 1996 John Wiley & Sons, Inc.     

Picodroplet-deposition of enzymes on functionalized self-assembled monolayers as a basis for miniaturized multi-sensor structures

We are reporting on a novel approach for structured immobilisation of enzymes on gold surfaces modified with monolayers of functionalised alkylthiols. The formation of enzyme spots is achieved by shooting very small volumes of an appropriate enzyme solution (down to 100 pl) onto a thiol-monolayer modified gold surface using a micro-dispenser. Formation of enzyme patterns is obtained by moving the micro-dispenser relative to the modified gold surface using a micro-positioning device. Enzyme spots with typical lateral dimensions of 100 μm are obtained, but also, more complex structures, e.g. lines or meander structures, can be achieved by multiple droplets dispensed during the concomitant movement of the micro-dispenser. The first enzyme layer on top of the functionalised thiol-monolayer is subsequently covalently immobilised using either carbodiimide activation of carboxilic headgroups at the enzyme or via already introduced activated ester functions at the monolayer. Immobilised enzyme activities of glucose oxidase and lactate oxidase patterns have been characterised by means of scanning electrochemical microscopy. The product of the enzyme-catalysed reaction, H₂O₂, is detected with an micro-electrode in the presence of either or both substrates, glucose and lactate, leading to a visualisation of the corresponding enzyme pattern and the lateral enzymatic activity.     

Thermodynamic studies on the adsorption of fibronectin adhesion-promoting peptide on nanothin films of poly(2-vinylpyridine) by SPR

This paper describes a methodology for preparing uniform, nanothin polymer films for the study of biomolecule adsorption by surface plasmon resonance spectroscopy (SPR). The methodology combines molecular self-assembly of alkanethiols on gold with surface-confined atom transfer radical polymerization (ATRP). Poly(2-vinylpyridine) was chosen to demonstrate the methodology, and growth kinetics were studied by ex situ ellipsometry. Atomic force microscopy (AFM) indicated that the polymer films were uniform with RMS roughness of approximately 0.5 nm. Subsequent SPR measurements were done to determine thermodynamic adsorption properties (deltaG, deltaH, and deltaS) between fibronectin adhesion-promoting peptide and the surface-confined poly(2-vinylpyridine) at 15, 20, and 25 degrees C. The flexibility in synthesis conditions and the opportunities for manipulating film thicknesses and graft densities that ATRP provides to grow polymer films from gold surfaces holds advantages over conventional spin-coating and grafting to approaches in the design of model polymer films for biomolecule adsorption studies. These advantages are described.     

Photocurable liquid biodegradable copolymers: in vitro hydrolytic degradation behaviors of photocured films of coumarin-endcapped poly(epsilon-caprolactone-co-trimethylene carbonate)

Coumarin-endcapped tetrabranched liquid copolymers composed of epsilon-caprolactone and trimethylene carbonate (TMC), prepared using pentaerythritol or four-branched poly(ethylene glycol) (PEG) as an initiator, were ultraviolet irradiated to produce photocured solid biodegradable copolymers. The hydrolytic degradation behaviors of photocured films were determined from the weight loss of the films. The initial hydrolysis rate (determined for up to 24 h using a quartz crystal microbalance) was enhanced with aqueous solutions of higher pH. The hydrolysis rate in the early period of immersion was increased with an increase in TMC content, whereas that in the later period (week order) decreased with a increase in TMC content. This inverse relation of composition dependence on the hydrolysis rate between the early and late periods was discussed. Topological measurements using scanning electron microscopy and atomic force microscopy as well as depth profiles of the fluorescein-stained hydrolyzed layer showed that for the pentaerythritol-initiated copolymer, irrespective of copolymer composition, hydrolysis occurred at surface regions and surface erosion proceeded with immersion time. For PEG-based copolymers, both surface erosion and bulk degradation occurred simultaneously. The hydrolyzed surfaces became highly wettable with water and exhibited noncell adhesivity.     

Layer-by-layer fabrication and direct electrochemistry of glucose oxidase on single wall carbon nanotubes

Layer-by-layer assembly of glucose oxidase (GOx) with single-wall carbon nanotubes (SWCNTs) is achieved on the electrode surface based on the electrostatic attraction between positively charged GOx in pH 3.8 buffer and negatively charged carboxylic groups of CNTs. The cyclic voltammetry and electrochemical impedance spectroscopy are used to characterize the formation of multilayer films. In deaerated buffer solutions, the cyclic voltammetry of the multilayer films of {GOx/CNT}_n shows two pairs of well-behaved redox peaks that are assigned to the redox reactions of CNTs and GOx, respectively, confirming the effective immobilization of GOx on CNTs using the layer-by-layer technique. The redox peak currents of GOx increase linearly with the increased number of layers indicating the uniform growth of GOx in multilayer films. The dependence of the cyclic voltammetric response of GOx in multilayer films on the scan rate and pH is also studied. A linear decrease of the reduction current of oxygen at the {GOx/CNT}-modified electrodes with the addition of glucose suggests that such multilayer films of GOx retain the bioactivity and can be used as reagentless glucose biosensors.     

Electrochemical properties of polyaniline/carboxydextran (PANI/carDEX) composite films for biofuel cells in neutral aqueous solutions

Electrochemical properties of composite films consisting of polyaniline/carboxydextran (PANI/carDEX) as a biofuel cell electrode platform were investigated. These composite films were formed on a planar gold surface through electropolymerization after a simple chemical modification of dextran with carboxyl groups. Cyclic voltammetry indicated that the composite films retained a redox activity in neutral pH environment. The PANI/carDEX composite films showed an electrocatalytic activity for the oxidation of ascorbic acid. The PANI/carDEX composite films also demonstrated an excellent electron-transfer mediating capability for the bioelectrocatalytic activation of glucose oxidase (GOx) toward the oxidation of glucose.     

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced byAlcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters, with 3-hydroxyvalerate (3HV) contents ranging from 17 to 60 mol%, were produced byAlcaligenes sp. MT-16, and their biocompatibility evaluated by the growth of Chinese hamster ovary (CHO) cells and the adsorption of blood proteins and platelets onto their film surfaces. The number of CHO cells that adhered to and grew on these films was higher with increasing 3HV content. In contrast, the tendency for blood proteins and platelets to adhere to the copolyester surfaces significantly decreased with increasing 3HV content. Examination of the surface morphology using atomic force microscopy revealed that the surface roughness was an important factor in determining the biocompatibility of theses copolyesters. The results obtained in this study suggest that poly(3HB-co-3HV) copolyesters, with >30 mol% 3HV, may be useful in biocompatible biomedical applications.     

Chitosan-mediated synthesis of gold nanoparticles on patterned poly(dimethylsiloxane) surfaces

Synthesis of gold nanoparticles on surfaces has been accomplished by the incubation of poly(dimethylsiloxane) (PDMS) films in tetrachloroauric(III) acid and chitosan solution at room temperature and 4 degrees C. One important point in the present study is that the synthesis selectively occurred on the PDMS surface. These observations are substantially different from the reaction in solution, in which no particles can be formed at room temperature. Computation of surface plasmon bands (SPBs) based on Mie theory suggests that the particles are partially coated by chitosan molecules, and the experimental results confirm the theoretical calculations. The proposed mechanism is that chitosan molecules adsorbed or printed on the PDMS surfaces act as reducing/stabilizing agents. Furthermore, PDMS films patterned with chitosan could induce localized synthesis of gold nanoparticles in regions capped with chitosan only. In this way, colloidal patterns were fabricated on the surfaces with high spatial selectivity simultaneously with the synthesis of the particles. Surface-induced fluorescence quenching was observed in the regions capped with gold nanoparticles as well.