Biomolecular sensors for neurotransmitter determination: electrochemical immobilization of glutamate oxidase at microelectrodes in a poly(o-phenylenediamine) film

The adsorption of glutamate oxidase onto 25 Μm and 10 Μm platinum microelectrodes followed by immobilization in an electrochemically polymerized non-conducting polymer, poly(o-phenylenediamine), is described as a method of fabricating an enzyme electrode for the amperometric determination of glutamate. The response of the enzyme electrodes were found to be highly reproducible with a linear dynamic range upto approximately 15 mmol dm^?3. The response of the 25 Μm and 10 Μm enzyme electrodes to glutamate were analysed using an established kinetic model and the potential application of the sensor for the study of neurotransmitter dynamics was investigated. The sensor was stable over a period of 30 days and the polymeric film was found to reduce interference from the electroactive compounds, uric acid and ascorbic acid.     

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.     

Electrochemical sensor for electrochemically inactive beta-D(+)-glucose using alpha-cyclodextrin template molecules

We report an electrochemical sensor for an electrochemically inactive organic compound using a self-assembled monolayer (SAM) formed on the gold surface from a solution of thiolated alpha-cyclodextrin (alpha-CD). The SAM makes up an array of ultramicroelectrodes, which capture electroactive molecules such as those of ferrocene. When this SAM-modified electrode is exposed to a solution containing an electrochemically inactive compound, e.g., glucose, the captured ferrocene molecules are replaced by the electroinactive molecules via an equilibrium established between the two compounds, lowering the current for ferrocene oxidation. The decrease in current is directly proportional to the amount of glucose added and the glucose can be analyzed up to approximately 0.80 mM, which is approximately 1/10 of a typical physiological concentration in blood serum. Formation constants of the surface-bound alpha-CD molecules with ferrocene and glucose are also reported.     

Wiring of enzymes to electrodes by ultrathin conductive polyion underlayers: enhanced catalytic response to hydrogen peroxide

Stable electroactive films were grown layer by layer on rough pyrolytic graphite electrodes featuring 4-nm underlayers of sulfonated polyaniline (SPAN) covered with a film containing myoglobin or horseradish peroxidase grown in alternating layers with poly(styrenesulfonate). The self-doped polyanionic SPAN layer, grown on a 2-nm polycation layer, was conductive between about 0.1 and -0.4 V vs SCE at pH 4.5. The enzyme films had the architecture PDDA/SPAN/(enzyme/PSS)3, where PDDA is poly(diallyldimethylammonium) ion. Comparisons of voltammetric measurements of electroactive protein with quartz crystal microbalance measurements of total protein showed that 90% or more of the protein was coupled to the electrode when the SPAN underlayer was present, as opposed to approximately 40% protein electroactivity when SPAN was absent. As a consequence of the highly efficient coupling between enzymes and electrode, the PDDA/SPAN/(enzyme/PSS)3 films exhibited a higher sensitivity for the electrochemical catalytic reduction of hydrogen peroxide. Amperometry at a rotating disk electrode at 0 V gave sensitivity for hydrogen peroxide up to 14 microA microM(-1) cm(-2) in the submicromolar concentration range and a detection limit of approximately 3 nM. Results suggest the future utility of ultrathin layers of conductive self-doping polyions in improving sensitivity of enzyme biosensors.     

Scanning tunneling microscopic and voltammetric studies of the surface structures of an electrochemically activated glassy carbon electrode

The microscopic surface structures of electrochemically activated glassy carbon have been examined by scanning tunneling microscopy. Experimental results demonstrate that there are two different types of electrode surface sites, corresponding to the bundles and bundle edges of the fibrous graphite microcrystallities. Electrochemical activation results in the formation of new void spaces of different sizes, and the structures are affected by the electrochemical activation procedures employed. The void volume resulting from cyclic polarization is usually smaller than that obtained by potentostatic activation. Electrochemical behaviors of the activated electrode are related to both the new void structures and the size of the electroactive species employed. On the basis of the STM voltammetric results, the microscopic structure of the activated electrode is proposed.     

聚吡咯膜电化学包埋固定酶及其复合酶生物传感器

采用电化学包埋法成功地将乳酸脱氢酶固定化在聚吡咯膜上,获得了具有生物活性和电活性的聚吡咯乳酸脱氢酶导电膜,并进一步制备了用以测定丙酮酸的聚吡咯乳酸脱氢酶金电极生物传感器。同时尝试用电化学包埋法将乳酸脱氢酶和辅酶NADH同时固定化在聚吡咯膜上,获得了具有电化学活性的聚吡咯乳酸脱氢酶NADH 金复合酶电极。深入的实验证实了复合电极生物传感器具有线性的工作曲线。     

Immobilization method for the preparation of biosensors based on pH shift-induced deposition of biomolecule-containing polymer films.

Miniaturization of amperometric biosensors is crucially dependent on the availability of methods for the nonmanual immobilization of biological recognition elements on the transducer surface. From an aqueous polymer suspension, the precipitation of a polymer film with entrapped biological recognition elements is initiated by electrochemically induced oxidation of H20 at the electrode surface. Using the locally generated H+ gradient, acidic side chains of the polymer are titrated, leading to a change in the polymer solubility and hence to the controlled deposition of a polymer film. To investigate the properties and limitations of this immobilization technology, the specific features of a glucose biosensor based on polymer-entrapped glucose oxidase and amperometric detection of enzymatically generated H202 were investigated. Besides the reproducibility of the immobilization procedure, the sensitivity (14.59 mA cm(-2) M(-1) at pH 7), long-term stability (up to 5000 measurements in a sequential-injection analyzer), dependence on enzyme concentration, polymer thickness, and possibilities to fabricate multilayer sensor architectures were exploited. In addition, the miniaturization potential of this nonmanual immobilization technology was evaluated by investigating the modification of microband electrode arrays and cross talk between the neighboring microsensors.     

An Enzyme Switch Employing Direct Electrochemical Communication between Horseradish Peroxidase and a Poly(aniline) Film

An enzyme switch, or microelectrochemical enzyme transistor, responsive to hydrogen peroxide was made by connecting two carbon band electrodes (～10 μm wide, 4.5 mm long separated by a 20-μm gap) with an anodically grown film of poly(aniline). Horseradish peroxidase (EC 1.11.1.7) was either adsorbed onto the poly(aniline) film or immobilized in an insulating poly(1,2-diaminobenzene) polymer grown electrochemically on top of the poly(aniline) film to complete the device. In the completed device, the conductivity of the poly(aniline) film changes from conducting (between - 0.05 and + 0.3 V vs SCE at pH 5) to insulating (>+0.3 V vs SCE at pH 5) on addition of hydrogen peroxide. The change in conductivity is brought about by oxidation of the poly(aniline) film by direct electrochemical communication between the enzyme and the conducting polymer. This was confirmed by measuring the potential of the poly(aniline) film during switching of the conductivity in the presence of hydrogen peroxide. The devices can be reused by rereducing the poly(aniline) electrochemically to a potential below +0.3 V vs SCE. A blind test showed that the device can be used to determine unknown concentrations of H(2)O(2) in solution and that, when used with hydrogen peroxide concentrations below 0.5 mmol dm(-)(3), the same device maybe reused several times. The possible development of devices of this type for use in applications requiring the measurement of low levels of hydrogen peroxide or horseradish peroxidase is discussed.     

Effect of ionic strength on the behavior of amperometric enzyme electrodes mediated by redox hydrogels

The electron-transfer behavior of electroactive hydrogels formed by cross-linking ferrocene poly(allylamine) (Fc-PAA) and glucose oxidase is investigated as a function of electrolyte ionic strength using several techniques. Cyclic voltammetry and electrochemical impedance spectroscopy show that the quantity cD(e)(1/2) increases with electrolyte concentration. Enhancement of enzyme catalysis for the oxidation of glucose mediated by Fc-PAA is also apparent at higher KNO(3) concentration. The electroactive redox center concentration, c, and the diffusion coefficient due to electron hopping in the gel, D(e), are independently measured by chronoamperometry at ultramicroelectrodes. Larger electrolyte ionic strength induces an increase in electroactive redox center concentration while D(e) slightly decreases. These results are rationalized in terms of the electrostatic interactions within the redox gel backbone due to water and ion exchange with the external electrolyte, producing swelling and shrinking of the hydrogel.     

Enzyme biosensor based on plasma-polymerized film-covered carbon nanotube layer grown directly on a flat substrate

We report a novel approach to fabrication of an amperometric biosensor with an enzyme, a plasma-polymerized film (PPF), and carbon nanotubes (CNTs). The CNTs were grown directly on an island-patterned Co/Ti/Cr layer on a glass substrate by microwave plasma enhanced chemical vapor deposition. The as-grown CNTs were subsequently treated by nitrogen plasma, which changed the surface from hydrophobic to hydrophilic in order to obtain an electrochemical contact between the CNTs and enzymes. A glucose oxidase (GOx) enzyme was then adsorbed onto the CNT surface and directly treated with acetonitrile plasma to overcoat the GOx layer with a PPF. This fabrication process provides a robust design of CNT-based enzyme biosensor, because of all processes are dry except the procedure for enzyme immobilization. The main novelty of the present methodology lies in the PPF and/or plasma processes. The optimized glucose biosensor revealed a high sensitivity of 38 μA mM(-1) cm(-2), a broad linear dynamic range of 0.25-19 mM (correlation coefficient of 0.994), selectivity toward an interferent (ascorbic acid), and a fast response time of 7 s. The background current was much smaller in magnitude than the current due to 10 mM glucose response. The low limit of detection was 34 μM (S/N = 3). All results strongly suggest that a plasma-polymerized process can provide a new platform for CNT-based biosensor design.     

Microfabricated electrophoresis chips for simultaneous bioassays of glucose, uric acid, ascorbic acid, and acetaminophen

A micromachined capillary electrophoresis chip is described for simultaneous measurements of glucose, ascorbic acid, acetaminophen, and uric acid. Fluid control is used to mix the sample and enzyme glucose oxidase (GOx). The enzymatic reaction, a catalyzed aerobic oxidation of glucose to gluconic acid and hydrogen peroxide, occurs along the separation channel. The enzymatically liberated neutral peroxide species is separated electrophoretically from the anionic uric and ascorbic acids in the separation/reaction channel. The three oxidizable species are detected at the downstream gold-coated thick-film amperometric detector at different migration times. Glucose can be detected within less than 100 s, and detection of all electroactive constituents is carried out within 4 min. Measurements of glucose in the presence of acetaminophen, a neutral compound, are accomplished by comparing the responses in the presence and absence of GOx in the running buffer. The reproducibility of the on-chip glucose measurements is improved greatly by using uric acid as an internal standard. Factors influencing the performance, including the GOx concentration, field strength, and detection potential, are optimized. Such coupling of enzymatic assays with electrophoretic separations on a microchip platform holds great promise for rapid testing of metabolites (such as glucose or lactate), as well as for the introduction of high-speed clinical microanalyzers based on multichannel chips.     

Biomolecular sensors for neurotransmitter determination: electrochemical immobilization of glutamate oxidase at microelectrodes in a poly(o-phenylenediamine) film

The adsorption of glutamate oxidase onto 25 m and 10 m platinum microelectrodes followed by immobilization in an electrochemically polymerized non-conducting polymer, poly(o-phenylenediamine), is described as a method of fabricating an enzyme electrode for the amperometric determination of glutamate. The response of the enzyme electrodes were found to be highly reproducible with a linear dynamic range upto approximately 15 mmol dm^–3. The response of the 25 m and 10 m enzyme electrodes to glutamate were analysed using an established kinetic model and the potential application of the sensor for the study of neurotransmitter dynamics was investigated. The sensor was stable over a period of 30 days and the polymeric film was found to reduce interference from the electroactive compounds, uric acid and ascorbic acid.     

Electrochemical and optical sugar sensors based on phenylboronic acid and its derivatives

Recent progress in electrochemical and optical sugar sensors based on phenylboronic acid (PBA) and its derivatives as recognition components is reviewed. PBAs are known to bind diol compounds including sugars to form cyclic boronate esters that are negatively charged as a result of the addition of OH^? ions from solution. Based on the formation of PBA charged species, sugars and their derivatives can be detected by means of electrochemical and optical techniques. For the development of PBA-based electrochemical sensing systems or sensors, PBA is modified with a redox-active marker, because PBA itself is electrochemically inactive, and ferrocene derivatives are often employed for this purpose. Ferrocene-modified PBAs have been used as redox-active additives in solution for the electrochemical detection of sugars and derivatives. PBA-modified electrodes have also been constructed as reagentless electrochemical sensors, where PBAs are immobilized on the surface of metal and carbon electrodes through mainly two routes: as a self-assembled monolayer film and as a polymer thin film. PBA-modified electrodes can be successfully used to detect sugars and derivatives through potentiometric and voltammetric responses. In addition, PBA-modified electrodes can be used for the immobilization of glycoenzymes on an electrode surface by the formation of boronate esters with carbohydrate chains in the glycoenzymes, thus resulting in enzyme biosensors. For the development of PBA-based optical sensors, a variety of chromophores and fluorophores have been coupled with PBA. Azobenzene dyes have been most frequently used for the preparation of colorimetric sugar sensors, in which the absorption wavelength and intensity of the dye are dependent on the type and concentration of added sugars. The sensitivity of the sensors is significantly improved based on multi-component systems in which alizalin red S, pyrocatechol violet, starch–iodine complex, and cyclodextrin are employed as indicators. Anthracene, pyranine, fluorescein, and rhodamine dyes have been used as fluorophores for fluorescence sensors. These dyes have been used in solution or immobilized in films, hydrogels, nanospheres, and quantum dots (QDs) to enhance the sensitivity. QDs-based sensors have been successfully applied for continuous monitoring of glucose in cells. Holographic glucose sensors have also been developed by combining PBA-immobilized hydrogels and photonic crystal colloidal arrays.     

Cell chip with nano-scale peptide layer to detect dopamine secretion from neuronal cells

A cell chip with a nano-scaled thin film of cysteine modified synthetic oligopeptide C(RGD)4 was fabricated to detect dopamine secretion from neuronal cells. Thin C(RGD)4 peptide layer was fabricated on chip surface for increasing the binding affinity of cells to gold electrode surface, which is essential for the electrochemical detection of dopamine released from PC12 cells. The structural formation of the peptide thin film was confirmed by both atomic force microscopy (AFM) and scanning electron microscopy (SEM). Redox characteristics of chemical dopamine were firstly characterized by voltammetric tool to compare the dopamine released from PC12 cells. Cells grown on the chip were then subjected to cyclic voltammetric (CV) analysis after 48 hours of incubation. The intensities of reduction peaks were found to be increased with increasing the concentrations of PC12 cells. In addition, the electrochemical redox signal increased more in the cells treated with glucose and potassium compared to the control group. Hence, the developed cell chip can be used to determine the effects of drugs on living cells electrochemically.     

Elimination of electrooxidizable interferant-produced currents in amperometric biosensors.

Electrooxidizable ascorbate, urate, and acetaminophen that interfere with amperometric glucose assays are completely and rapidly oxidized by hydrogen peroxide in a multilayer electrode. The multilayer electrode is composed of an immobilized, but not electrically "wired", horseradish peroxidase (HRP) film coated onto a film of electrically "wired" glucose oxidase (GO). The "wired" enzyme is connected by a redox epoxy network to a vitreous carbon electrode. The current from the electrooxidizable interferants is decreased by their peroxidase-catalyzed preoxidation by a factor of 2500, and the glucose/interferant current ratio is increased 10(3)-fold. Undesired electroreduction of hydrogen peroxide can result when HRP is also "wired" to the electrode. Such unwanted "wiring" is prevented by incorporating an electrically insulating barrier layer between the wired GO film and the HRP film. The hydrogen peroxide necessary for elimination of interferants can be added externally, or when this is not possible, it can be generated in situ by means of a coupled enzyme reaction.     

Electroosmotic flow and its contribution to iontophoretic delivery

Iontophoresis is the movement of charged molecules in solution under applied current using pulled multibarrel glass capillaries drawn to a sharp tip. The technique is generally nonquantitative, and to address this, we have characterized the ejection of charged and neutral species using carbon-fiber electrodes attached to iontophoretic barrels. Our results show that observed ejections are due to the sum of iontophoretic and electroosmotic forces. With the use of the neutral, electroactive molecule 2-(4-nitrophenoxy) ethanol (NPE), which is only transported by electroosmotic flow (EOF), a positive correlation between the amount ejected and the diameter of each barrel's tip was found. In addition, using various charged and neutral electroactive compounds we found that, when each compound is paired with the EOF marker, the percentage of the ejection due to EOF remains constant. This percentage varies for each pair of compounds, and the differences in mobility are positively correlated to differences in electrophoretic mobility. Overall, the results show that capillary electrophoresis (CE) can be used to predict the percentage of ejection that will be due to EOF. With this information, quantitative iontophoresis is possible for electrochemically inactive drugs by using NPE as a marker for EOF.     

Application of electrochemically reduced graphene oxide on screen-printed ion-selective electrode

In this study, a novel disposable all-solid-state ion-selective electrode using graphene as the ion-to-electron transducer was developed. The graphene film was prepared on screen-printed electrode directly from the graphene oxide dispersion by a one-step electrodeposition technique. Cyclic voltammetry and electrochemical impedance spectroscopy were employed to demonstrate the large double layer capacitance and fast charge transfer of the graphene film modified electrode. On the basis of these excellent properties, an all-solid-state calcium ion-selective electrode as the model was constructed using the calcium ion-selective membrane and graphene film modified electrode. The mechanism about the graphene promoting the ion-to-electron transformation was investigated in detail. The disposable electrode exhibited a Nernstian slope (29.1 mV/decade), low detection limit (10(-5.8) M), and fast response time (less than 10 s). With the high hydrophobic character of graphene materials, no water film was formed between the ion-selective membrane and the underlying graphene layer. Further studies revealed that the developed electrode was insensitive to light, oxygen, and redox species. The use of the disposable electrode for real sample analysis obtained satisfactory results, which made it a promising alternative in routine sensing applications.     

Construction of amperometric immunosensors based on the electrogeneration of a permeable biotinylated polypyrrole film

The construction of amperometric immunosensors to cholera antitoxin immunoglobulins were shown to have improved sensitivity when the cholera toxin B subunit biorecognition entity was linked to an electrogenerated biotinylated polypyrrole film copolymerized with pyrrole-lactobionamide monomer. The copolymer exhibits greater film permeability than biotinylated polypyrrolic or polyphenolic films for the permeation of electroactive species. Hence, when the presence of the HRP marker of the immunoassay was determined using hydroquinone, the production of electroactive quinone was shown to permeate faster to the electrode, thus providing a faster response time.     

Catalysis-electrochemical determination of zeptomole enzyme and its application for single-cell analysis

A novel electrochemical method for determination of zeptomole amounts of enzyme was developed by a combination of on-capillary enzyme-catalyzed reaction and electrochemical detection. A limit of detection (LOD) of zeptomole (zmol, 10(-)(21) mol) was achieved by monitoring the product of the enzyme-catalyzed reaction. In this method, after enzyme molecules were electrokinetically injected into the capillary, they were electromigrated to the section of the capillary immersed in a warm water bath, where the enzyme molecules reacted with the enzyme substrates in the running buffer in the presence of the activator of the enzyme-catalyzed reaction. Then the electroactive product zone of the enzyme-catalyzed reaction was electromigrated to the horn-shaped outlet of the capillary and electrochemically detected by a carbon fiber disk bundle electrode at a constant potential. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as the model enzyme. A LOD of 1.3 zmol was achieved. This method was applied to determine zeptomoles of G6PDH in individual human erythrocytes.     

Glucose and lactate biosensors based on redox polymer/oxidoreductase nanocomposite thin films

Glucose and lactate enzyme electrodes have been fabricated through the deposition of an anionic self-assembled monolayer and subsequent redox polymer/enzyme electrostatic complexation on gold substrates. These surfaces were functionalized with a negative charge using 11-mercaptoundecanoic acid (MUA), followed by alternating immersions in cationic redox polymer solutions and anionic glucose oxidase (GOX) or lactate oxidase (LAX) solutions to build the nanocomposite structure. The presence of the multilayer structure was verified by ellipsometry and sensor function characterized electrochemically. Reproducible analyte response curves from 2 to 20 mM (GOX) and 2-10 mM (LAX) were generated with the standard deviation between multiple sensors between 12 and 17%, a direct result of the reproducibility of the fabrication technique. In the case of glucose enzyme electrodes, the multilayer structure was further stabilized through the introduction of covalent bonds within and between the layers. Chemical cross-linking was accomplished by exposing the thin film to glutaraldehyde vapors, inducing linkage formation between lysine and arginine residues present on the enzyme periphery with amine groups present on a novel redox polymer, poly[vinylpyridine Os(bisbipyridine)2Cl]-co-allylamine. Finally, an initial demonstration of thin-film patterning was performed as a precursor to the development of redundant sensor arrays. Microcontact printing was used to functionalize portions of a gold surface with a blocking agent, typically 1-hexadecanethiol. This was followed by immersion in MUA to functionalize the remaining portions of gold with negative charges. The multilayer deposition process was then followed, resulting in growth only on the regions containing MUA, resulting in a "positive"-type pattern. This technique may be used for fabrication of thin-film redundant sensor arrays, with thickness under 100 angstrom and lateral dimensions on a micrometer scale.