Characterization of enzyme immobilized carbon electrode using covalent-entrapment with polypyrrole

ABSTRACT

This study aimed to improve the enzyme immobilization of electrodes to increase electrical power and lifespan using glucose oxidase in a bioanode, laccase in a biocathode, and glucose as a fuel. Both of these enzymes were immobilized on a carbon paper through covalent entrapment. Electrodes were characterized using electrochemical measurements (cyclic voltammetry) and enzymatic biofuel cells. For continuous 16 h, the maximum power density achieved for a hydrophobic electrode was approximately 80 μW/cm² at 0.13 V. For a hydrophilic electrode, the yield was approximately 130 μW/cm² at 0.25 V, which was significantly higher than that for the hydrophobic electrode. The measurements were performed at a working temperature of 37°C with phosphate buffer solution of pH 7 as an electrolyte, and 10 mM glucose was added to the anode as a fuel. The hydrophilic electrode was superior to the hydrophobic electrode because of the covalent entrapment immobilization of glucose oxidase and laccase enzymes in enzymatic biofuel cells.     

Nanothin ferrocene film plasma polymerized over physisorbed glucose oxidase: high-throughput fabrication of bioelectronic devices without chemical modifications

We describe a method for creating a mediator-containing interface between an enzyme and an electrode, achieving simpler and more reliable immobilization of the enzyme with the enhanced detection sensitivity. A nanothin polymer film containing a redox mediator, made of dimethylaminomethylferrocene, was plasma-deposited directly onto a glucose oxidase-physisorbed electrode, with which a relevant bioelectrochemical signal was observed without prior or further chemical modification of the enzyme molecules. The results of the surface characterizations before and after the enzyme immobilization showed that this method gave control over the spatial orientation of single enzyme molecules in favor of efficient and reproducible signal generation. Considering that the film deposition was performed using microfabrication-compatible organic plasma, our new method has a great potential of enabling high-throughput production of bioelectronic devices without chemical modification steps.     

Voltammetric and reference microelectrodes with integrated microchannels for flow through microvoltammetry. 1. The microcell

This paper describes the construction of 2 microsensor units for on-line voltammetric detection inside a cylindrical microcell (a working microsensor unit and a reference and auxiliary microsensor unit), for application to heavy-metal analysis in complex media such as natural waters. Both microsensor units include a channel for the solution renewal in the microcell after analysis. The working microsensor, a Hg-plated Ir microelectrode, is protected against fouling with an agarose gel including a hydrophobic chromatographic phase (C18). The fabrication steps and the quality tests related to long-term use and reliability, as well as to precision, are described. The application of the protective gel layer against fouling by hydrophobic or surface active small molecules is of general application, reliable, and very efficient. The reference and auxiliary unit is composed by an iridium oxide based mini reference electrode, an auxiliary Pt electrode, and a circulation channel. It is built to enable its use inside a 700-microm-diameter tubing connected to a hollow fiber supported liquid membrane lumen (volume, 5-10 microL) for heavy-metal analysis. However, it can be used in any other microanalytical system. The reference electrode is sufficiently stable for voltammetric applications (1-2 mV drift/day), and its lifetime is more than one year. The Ti/IrO2 core is immersed in a pH-buffered agarose gel, to guarantee potential stability even when the electrode is immersed in variable pH solutions.     

Immunobiosensor chips for detection of Escherichia coil O157:H7 using electrochemical impedance spectroscopy

Impedance biosensor chips were developed for detection of Escherichia coli O157:H7 based on the surface immobilization of affinity-purified antibodies onto indium tin oxide (ITO) electrode chips. The immobilization of antibodies onto ITO chips was carried out using an epoxysilane monolayer to serve as a template for chemical anchoring of antibodies. The surface characteristics of chips before and after the binding reaction between the antibodies and antigens were characterized by atomic force microscopy (AFM). The patterns of the epoxysilanes monolayer, antibodies, and E. coli cells were clearly observed from the AFM images. Alkaline phosphatase as the labeled enzyme to anti-E. coli O157:H7 antibody was used to amplify the binding reaction of antibody-antigen on the chips. The biocatalyzed precipitation of 5-bromo-4-chloro-3-indolyl phosphate by alkaline phosphatase on the chips in pH 10 PBS buffer containing 0.1 M MgCl2 increased the electron-transfer resistance for a redox probe of Fe(CN)6(3-/4-) at the electrode-solution interface or the electrode resistance itself. Electrochemical impedance spectroscopy and cyclic voltammetric method were employed to follow the stepwise assembly of the systems and the electronic transduction for the detection of E. coli. The biosensor could detect the target bacteria with a detection limit of 6 x 10(3) cells/mL. A linear response in the electron-transfer resistance for the concentration of E. coli cells was found between 6 x 10(4) and 6 x 10(7) cells/mL.     

A strategy for constructing a hybrid bilayer membrane based on a carbon substrate

We construct a hybrid bilayer membrane (HBM) on a new substrate-carbon electrode. It is an extension of HBM based on other substrates. Primary alkylamine was chemically modified onto the surface of a carbon electrode by electrochemical scans; thus, a monolayer was formed on the electrode. Because the alkane chains section is toward the outside, a hydrophobic surface was constructed. Then a lipid monolayer was spread on the hydrophobic surface of the carbon electrode. The formed HBM was characterized by electrochemical and ATR-FT-IR methods. From ATR-FT-IR results, the lipid order parameter (S) of 0.73 was obtained. This kind of hybrid membrane has the advantages of a lipid/alkanethiol HBM. A potential application of this HBM as a biosensor (detecting K+) was given.     

Towards nanomedicine with a supramolecular approach: a review

A review dedicated mainly to the results obtained by the authors on the use of cyclodextrin (CD) derivatives on protein (enzyme) stabilization through covalent and non-covalent interactions (host-guest supramolecular interactions) is presented here. This latter procedure served to introduce a new method for enzyme immobilization on metallic surfaces that can be used to prepare biosensors and therapeutic nanodevices. The surfaces of gold (and silver) electrodes and nanoparticles were modified with sulphur-containing cyclodextrin derivatives. The protein (enzyme) was then supramolecularly immobilized on the modified surface when one or more of its bulky hydrophobic moieties was included into the CD cavity. The protein can also be modified with a typical CD guest, such as adamantane, to achieve a more stable immobilization. Different examples are presented, such as a biosensor based on monolayers of adamantane-modified cytochrome c and a bienzymatic nanodevice comprising gold nanoparticles stabilized with CD associated to catalase and superoxide dismutase modified with complementary host-guest residues. The possibilities of this new approach for the development of biosensors and therapeutic nanodevices are analyzed.     

Enzyme-modulated cleavage of dsDNA for supramolecular design of biosensors.

Supramolecular docking and immobilization of biotinylated dsDNA onto a self-assembled monolayer of avidin have been measured using impedance spectroscopy and quartz crystal microbalance technique. The formation of the serial assembly was first achieved by linearizing circular plasmid dsDNA using BamH I endonuclease enzyme. This was followed by a bisulfite-catalyzed transamination reaction in order to biotinylate the dsDNA. The reaction is single-strand specific, and it specifically targets unpaired cytosine bases generated during the enzyme cleavage. The biotinylated dsDNA was then used as a ligand at a gold electrode containing avidin. The process was monitored by ac impedance spectroscopy that was used to probe the changes in interfacial electron-transfer resistance upon binding and a microgravimetric quartz crystal microbalance that reflected in situ mass changes on the dsDNA-functionalized substrates. Our results demonstrated that this approach could be employed for the determination of small-molecular-weight organics such as cisplatin, daunomycin, bisphenol A, chlorinated phenols, and ethidium bromide. A detection limit in the magnitude of ca. 10 nM was achieved. This immobilization technique provides a generic approach for dsDNA-based sensor development and for the monitoring of DNA-analyte interactions.     

Amperometric detection of Escherichia coli heat-labile enterotoxin by redox diacetylenic vesicles on a sol-gel thin-film electrode

Supramolecular assemblies (bilayer vesicles) prepared from ferrocenic diacetylene lipid and the cell surface receptor ganglioside GM1 are utilized to construct an amperometric biosensor for Escherichia coli heat-labile enterotoxin on a sol-gel thin-film electrode. The bilayer vesicles adsorbed on the sol-gel film provide an open platform for molecular recognition, while the electrochemical communication between the incorporated redox lipids and the electrode is influenced by the binding of the toxin. Cyclic voltammetric studies suggest a facile redox reaction for the adsorbed supramolecular assembly, which allows the sensor to detect enterotoxin up to 3 ppm (3.6 x 10(-8) M) concentration. The apparent diffusion coefficients for the redox lipids in the assembly were observed to be in the range of 4.73 x 10(-8) -2.30 x 10(-8) cm/s2. A mechanism of lateral electron transport of redox lipids controlled by biomolecular recognition is proposed.     

Impedance sensing of DNA binding drugs using gold substrates modified with gold nanoparticles

Interfacial interactions between immobilized DNA probes and DNA-specific sequence binding drugs were investigated using impedance spectroscopy toward the development of a novel biosensing scheme. The impedance measurements are based on the charge-transfer kinetics of the [Fe(CN)6]3-/4- redox couple. Compared to bare gold surfaces, the immobilization of DNA and then the DNA-drug interaction on electrode surfaces altered the capacitance and the interfacial electron resistance and thus diminished the charge-transfer kinetics by reducing the active area of the electrode or by preventing the redox species from approaching the electrode. Electrochemical deposition of gold nanoparticles on a gold electrode surface showed significant improvement in sensitivity. DNA-capped gold nanoparticles on electrodes act as selective sensing interfaces with tunable sensitivity due to higher amounts of DNA probes and the concentric orientation of the DNA self-assembled monolayer. The specificity of the interactions of two classical minor groove binders, mythramycin, a G-C specific-DNA binding anticancer drug, netropsin, an A-T specific-DNA binding drug and an intercalator, nogalamycin on AT-rich DNA-modified substrate and GC-rich DNA-modified substrate are compared. Using gold nanoparticle-deposited substrates, impedance spectroscopy resulted in a 20-40-fold increase in the detection limit. Arrays of deposited gold nanoparticles on gold electrodes offered a convenient tool to subtly control probe immobilization to ensure suitably adsorbed DNA orientation and accessibility of other binding molecules.     

Amplification of amperometric biosensor responses by electrochemical substrate recycling. 3. Theoretical and experimental study of the phenol-polyphenol oxidase system immobilized in Laponite hydrogels and layer-by-layer self-assembled structures

The amperometric response toward phenol of PPO-based rotating disk bioelectrodes is analyzed on the basis of a kinetic model taking into account internal and external mass transport effects and a CEC' electroenzymatic mechanism. Monophenolase activity of PPO catalyses the oxidation of phenol to o-quinone (step C). o-Quinone can then enter an amplification recycling process involving electrochemical reduction (step E) and enzymatic reoxidation (step C': catecholase activity). The rate-limiting steps such as monophenolase activity, catecholase recycling, permeability of the membrane, and activity and accessibility of the catalytic enzyme sites are theoretically considered and experimentally demonstrated for different electrode configurations including PPO immobilized in Laponite hydrogels and layer-by-layer self-assembled multilayers of PPO and poly(diallyldimethylammonium).     

Tailoring mesoporous-silica nanoparticles for robust immobilization of lipase and biocatalysis

The rational design of nano-carriers is critical for modem enzyme immobilization for advanced biocatalysis.Herein,we report the synthesis of octadecylalkylmodified mesoporous-silica nanoparticles (C18-MSNs) with a high C18 content (～19 wt.％) and tunable pore sizes (1.6-13 nm).It is demonstrated that the increased hydrophobic content and a tailored pore size (slightly larger than the size of lipase) are responsible for the high performance of immobilized lipase.The optimized C18-MSNs exhibit a loading capacity of 711 mg/g and a specific activity 5.23 times higher than that of the free enzyme.Additionally,93％ of the initial activity is retained after reuse five times,which is better than the best performance reported to date.Our findings pave the way for the robust immobilization of lipase for biocatalytic applications.     

Nanoporous alkoxy-derived titanium oxide coating: a reactive overlayer for functionalizing titanium surface

Effective immobilization of bioactive substances such as adhesive proteins, synthetic peptides and growth factors on metallic substrates is required for a number of medical applications. In the present work, evidence is presented to show that an alkoxy-derived nano-porous titanium oxide coating, synthesized electrochemically on titanium in methanolic electrolytes, may act as an effective interface for functionalizing a titanium surface. It is demonstrated that nanoporous oxide coatings could facilitate fast diffusion of small organic molecules within the oxide network and form strong chemical bonds with the functional groups of these molecules at room temperature. Fourier transformed–infrared spectroscopy was used to investigate the nature of the interfacial interactions between the oxide network and a range of molecules containing COOH, OH, NH2, C=O and phosphoric acid functional groups. The results indicate that the nanometre-sized oxide clusters within the coating may play an essential role in effective immobilization of organic molecules by providing numerous binding sites for chemisorption of these species. The surface-derivatized oxide coating may provide a solid phase for the subsequent attachment of a broad range of biochemically active molecules on the titanium surface. © 1998 Chapman & Hall     

Vertically aligned carbon nanotube probes for monitoring blood cholesterol

Detection of blood cholesterol is of great clinical significance. The amperometric detection technique was used for the enzymatic assay of total cholesterol. Multiwall carbon nanotubes?(MWNTs), vertically aligned on a silicon platform, promote heterogeneous electron transfer between the enzyme and the working electrode. Surface modification of the MWNT with a biocompatible polymer, polyvinyl alcohol?(PVA), converted the hydrophobic nanotube surface into a highly hydrophilic one, which facilitates efficient attachment of biomolecules. The fabricated working electrodes showed a linear relationship between cholesterol concentration and the output signal. The efficacy of the multiwall carbon nanotubes in promoting heterogeneous electron transfer was evident by distinct electrochemical peaks and higher signal-to-noise ratio as compared to the Au electrode with identical enzyme immobilization protocol. The selectivity of the cholesterol sensor in the presence of common interferents present in human blood, e.g.?uric?acid, ascorbic acid and glucose, is also reported.     

Phospholipid-sepiolite biomimetic interfaces for the immobilization of enzymes

Biomimetic interfaces based on phosphatidylcholine (PC) assembled to the natural silicate sepiolite were prepared for the stable immobilization of the urease and cholesterol oxidase enzymes. This is an important issue in practical advanced applications such as biocatalysis or biosensing. The supported lipid bilayer (BL-PC), prepared from PC adsorption, was used for immobilization of enzymes and the resulting biomimetic systems were compared to several other supported layers including a lipid monolayer (ML-PC), a mixed phosphatidylcholine/octyl-galactoside layer (PC-OGal), a cetyltrimethylammonium monolayer (CTA), and also to the bare sepiolite surface. Interfacial characteristics of these layers were investigated with a focus on layer packing density, hydrophilicity/hydrophobicity, and surface charge, which are being considered as key points for enzyme immobilization and stabilization of their biological activity. Cytoplasmic urease and membrane-bound cholesterol oxidase, which served as model enzymes, were immobilized on the different PC-based hybrid materials to probe their biomimetic character. Enzymatic activity was assessed by cyclic voltammetry and UV-vis spectrophotometry. The resulting enzyme/bio-organoclay hybrids were applied as active phase of a voltammetric urea biosensor and cholesterol bioreactor, respectively. Urease supported on sepiolite/BL-PC proved to maintain its enzymatic activity over several months while immobilized cholesterol oxidase demonstrated high reusability as biocatalyst. The results emphasize the good preservation of bioactivity due to the accommodation of the enzymatic system within the biomimetic lipid interface on sepiolite.     

Biosensors based on membrane-bound enzymes immobilized in a 5-(octyldithio)-2-nitrobenzoic acid layer on gold electrodes

Gold electrodes were modified through chemisorption of 5-(octyldithio)-2-nitrobenzoic acid (ODTNB). ODTNB includes a long chain in a short-length thio acid, providing a heterogeneous-like alkanethiol layer after adsorption on gold electrodes. Membrane-bound enzymes, in particular D-fructose dehydrogenase (FDH), D-gluconate dehydrogenase (GADH), and L-lactic dehydrogenase (cytochrome b2) (Cyb2), were immobilized onto ODTNB-modified gold electrodes simply by adsorption. The short-length thio acid may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate enables hydrophobic interaction with the largely lipophilic, membrane-bound enzymes. The immobilization of FDH, GADH, and Cyb2 onto ODTNB-modified gold surfaces has been studied with the quartz crystal microbalance (QCM). Spectrophotometric and electrochemical assays indicate that the immobilized enzyme retains its enzymatic activity after immobilization onto the ODTNB-modified gold surface. The amount of immobilized (and active) enzyme was estimated from QCM to be of the order of 2.5 x 10(-12)-5.3 x 10(-12) mol x cm(-2). A fructose biosensor was developed, making use of a gold surface modified with ODTNB and fructose dehydrogenase, employing hydroxymethylferrocene as a mediator in solution. Calibration curves exhibited a linear relation between the biosensor response and the substrate concentration up to 0.7 mM. Statistical analysis gave an excellent linear correlation (r = 0.9993) and a sensitivity of 6.1 mM(-1) fructose. The biosensor shows a significant stable catalytic current for at least 25 days.     

An amperometric fructose biosensor based on fructose dehydrogenase immobilized in a membrane mimetic layer on gold

A prototype amperometric fructose biosensor based on membrane-bound fructose dehydrogenase (Gluconobacter sp.) and the coenzyme ubiquinone-6 immobilized in a membrane mimetic layer on a gold electrode has been constructed and tested. A bare gold electrode first was modified through chemisorption of a mixture of octadecyl mercaptan and two short-chain disulfides, 3,3'-dithiodipropionic acid and cystamine dihydrochloride. The membrane-bound enzyme, coenzyme, and additional phospholipid were codeposited through a detergent dialysis protocol. The short-chain modifiers may provide electrostatic interactions with enzyme surface charges, while the alkanethiolate and phospholipids enable hydrophobic interaction with the largely lipophilic, membrane-bound enzyme. At oxidizing potentials, the enzyme electrode responded with catalytic current densities up to 45 μA/cm(2) when exposed to fructose at 10 mM. The sensor exhibited a response time of less than 20 s, a sensitivity of 15 μA/cm(2)·mM and a detection limit of less than 10 μM. Biosensor measurements of d-fructose in apple and orange juice agreed to within a few percent with those made with an enzymatic spectrophotometric assay. The membrane mimetic layer effectively blocked access of interfering ascorbic acid to the electrode surface. Only a 4% positive error was observed in the presence of ascorbic acid at 5% of the fructose concentration (2 mM), which indicates that this construct could be particularly useful for quantitation of fructose in citrus juice.     

Liposome Crosslinked Polyacrylamide/DNA Hydrogel: a Smart Controlled‐Release System for Small Molecular Payloads

A novel stimuli‐responsive hydrogel system with liposomes serving as both noncovalent crosslinkers and functional small molecules carriers for controlled‐release is developed. Liposomes can crosslink polyacrylamide copolymers functionalized with cholesterol‐modified DNA motifs to yield a DNA hydrogel system, due to the hydrophobic interaction between cholesteryl groups and the lipid bilayer of liposomes. Functional information encoded DNA motifs on the polymer backbones endow the hydrogel with programmable smart responsive properties. In a model system, the hydrogel exhibits stimuli‐responsive gel‐to‐sol transformation triggered by the opening of DNA motifs upon the presence of a restriction endonuclease enzyme, EcoR I, or temperature change, realizing the controlled‐release of liposomes which are highly efficient carriers of active small molecules payloads. Two active molecules, 1,1‐dioctadecyl‐3,3,3,3‐tetramethylindodicarbocyanine perchlorate (DiIC18(5)) and calcein, are chosen as the hydrophobic and hydrophilic model payloads, respectively, to address the feasibility of the releasing strategy. Moreover, the hydrogel exhibits injectable property as well as self‐recovery behaviors.     

La_(0.7)Sr_(0.3)NiO_3/壳聚糖/聚吡咯/牛血红蛋白修饰玻碳电极的制备及构建过氧化氢传感器

在玻碳电极上修饰一层表面均匀的聚吡咯膜,然后利用钙钛矿和壳聚糖的复合膜来固定牛血红蛋白(Hb),制备出性能良好的过氧化氢生物传感器.采用循环伏安(CV)和扫描电镜对修饰电极进行了表征.该传感器对H2O2具有好的催化响应,且响应快.在优化的实验条件下,所制备的传感器对H2O2的线性范围为7.0×10-6～1.5×10-3mol/L,检测限为9.0×10-8mol/L.     

Differential impedance spectroscopy for monitoring protein immobilization and antibody-antigen reactions

This work describes the theoretical and experimental approaches for monitoring the interfacial biomolecular reaction between immobilized antibody and the antigen binding partner using novel differential impedance spectroscopy. The prerequisite of any biosensor is the immobilization of macromolecules onto the surface of a transducer. It is clear that the function of most macromolecules changes from what is observed in solution once immobilization has occurred. In the worst case, molecules entirely lose their binding activity almost immediately after immobilization. Certain conditions (e.g., denaturation, interfacial effects based on ionic strength, surface charge, dielectric constants, etc.) at interfaces are responsible for alterations of binding activity; it is not clear whether a combination of such processes is understood. However, these processes in combination must be reliably modeled in order to predict the outcome for most macromolecules. This work presents the theoretical and practical means for elucidating the surface reactivity of biomolecular reagents using ion displacement model with antibody-antigen (Ab-Ag) reaction as the test case. The Ab-Ag reaction was directly monitored using a dual-channeled, impedance analyzer capable of 1 measurement/s using covalent immobilization chemistry and polymer-modified electrodes in the absence of a redox probe. The evidence of Ab-Ag binding was revealed through the evolution of differential admittance. The surface loading obtained using the covalent immobilization chemistry was 9.0 x 10(16)/cm2, whereas with polymer-modified electrodes, the surface loading was 9.0 x 10(15)/cm2, representing a 10 times increase in surface reactivity. The proposed approach may be applicable to monitoring other surface interfacial reactions such as DNA-DNA interactions, DNA-protein interactions, and DNA-small molecule interactions.     

Electrogenerated chemiluminescence. 67. Dependence of light emission of the tris(2,2')bipyridylruthenium(II)/tripropylamine system on electrode surface hydrophobicity

We describe the effect of electrode surface hydrophobicity on the electrochemical behavior and electrogenerated chemiluminescence (ECL) of Ru(bpy)3(2+) (bpy = 2,2'-bipyridyl)/tripropylamine (TPrA) system. Gold and platinum electrodes were modified with different thiol monolayers. The hydrophobicity of the electrode surfaces changed with different terminal groups of the thiol molecules. The oxidation rate of TPrA was found to be much larger at the modified electrode with a more hydrophobic surface. The adsorption of neutral TPrA species on this kind of surface was assumed to contribute to the faster anodic kinetics. Due to the rapid generation of the highly reducing radical, TPrA., ECL intensity increased significantly at more hydrophobic electrodes. This electrode surface effect in the ECL analytical system allows one to improve the detection sensitivity at low concentrations of Ru(bpy)3(2+). The surfactant effect on the ECL process was also examined and discussed based on the change of electrode hydrophobicity by the adsorption of surfactant species.