Functionalization of a poly(amidoamine) dendrimer with ferrocenyls and its application to the construction of a reagentless enzyme electrode

Poly(amidoamine) dendrimers having various degrees of modification with the redox-active ferrocenyls were prepared by controlling the molar ratio of ferrocenecarboxaldehyde to amine groups of dendrimers. By alternate layer-by-layer depositions of partial ferrocenyl-tethered dendrimers (Fc-D) with periodate-oxidized glucose oxidase (GOx) on a Au surface, an electrochemically and enzymatically active multilayered assembly of enzyme was constructed. The resulting GOx/Fc-D multilayer-associated electrodes were electrochemically analyzed, and the surface concentration of ferrocenyl groups, active enzyme coverage, and sensitivity were estimated. A 32% dendrimer modification level of surface amines to ferrocenyls was found to be an optimum in terms of enzyme-dendrimer network formation, electrochemical interconnectivity of ferrocenyls, and electrode sensitivity. With the prepared Fc(32%)-tethered dendrimers, mono- and multilayered GOx/Fc-D electrodes were constructed, and their electrochemical and catalytic properties were characterized. The bioelectrocatalytic signals from the multilayered GOx/Fc-D electrodes were shown to be directly correlated to the number of deposited bilayers. From this result, it seems that the electrode sensitivity is directly controllable, and the multilayer-forming strategy with partial ferrocenyl-tethered dendrimers is useful for the construction of reagentless biosensors.     

Following the biocatalytic activities of glucose oxidase by electrochemically cross-linked enzyme-Pt nanoparticles composite electrodes

An integrated platinum nanoparticles (NPs)/glucose oxidase (GOx) composite film associated with a Au electrode is used to follow the biocatalytic activities of the enzyme. The film is assembled on a Au electrode by the electropolymerization of thioaniline-functionalized Pt NPs and thioaniline-modified GOx. The resulting enzyme/Pt NPs-functionalized electrode stimulates the O 2 oxidation of glucose to gluconic acid and H 2O 2. The modified electrode is then implemented to follow the activity of the enzyme by the electrochemical monitoring of the generated H 2O 2. The effect of the composition of the Pt NPs/GOx cross-linked nanostructures and the optimal conditions for the preparation of the electrodes are discussed.     

Modified gold surfaces by 6-(ferrocenyl)hexanethiol/dendrimer/gold nanoparticles as a platform for the mediated biosensing applications

An electrochemical biosensor mediated by using 6-(Ferrocenyl) hexanethiol (FcSH) was fabricated by construction of gold nanoparticles (AuNPs) on the surface of polyamidoamine dendrimer (PAMAM) modified gold electrode. Glucose oxidase (GOx) was used as a model enzyme and was immobilized onto the gold surface forming a self assembled monolayer via FcSH and cysteamine. Cyclic voltammetry and amperometry were used for the characterization of electrochemical response towards glucose substrate. Following the optimization of medium pH, enzyme loading, AuNP and FcSH amount, the linear range for the glucose was studied and found as 1.0 to 5.0 mM with the detection limit (LOD) of 0.6 mM according to S/N = 3. Finally, the proposed Au/AuNP/(FcSH + Cyst)/PAMAM/GOx biosensor was successfully applied for the glucose analysis in beverages, and the results were compared with those obtained by HPLC.     

Multilayer membranes via layer-by-layer deposition of glucose oxidase and Au nanoparticles on a Pt electrode for glucose sensing

We prepared multilayer membranes by the layer-by-layer deposition of glucose oxidase (GOx) and Au nanoparticles (5, 10, or 50 nm φ) on sensor substrates, such as a Pt electrode and a quartz glass plate, to prepare glucose sensors. The enzyme activity of GOx remained even in alternate assemblies, and the activity increased with the increasing number of depositions. The apparent K_m values of the deposited GOx were 28–32 mM, while a reported value in a solution is 33 mM. These results suggest that Au nanoparticles can be used as binders for the deposition of GOx without significant change in the affinity between GOx and glucose.     

Fabrication of a glucose biosensor based on citric acid assisted cobalt ferrite magnetic nanoparticles

A novel and practical glucose biosensor was fabricated with immobilization of Glucose oxidase (GOx) enzyme on the surface of citric acid (CA) assisted cobalt ferrite (CF) magnetic nanoparticles (MNPs). This innovative sensor was constructed with glassy carbon electrode which is represented as (GOx)/CA-CF/(GCE). An explicit high negative zeta potential value (-22.4 mV at pH 7.0) was observed on the surface of CA-CF MNPs. Our sensor works on the principle of detection of H2O2 which is produced by the enzymatic oxidation of glucose to gluconic acid. This sensor has tremendous potential for application in glucose biosensing due to the higher sensitivity 2.5 microA/cm2-mM and substantial increment of the anodic peak current from 0.2 microA to 10.5 microA.     

PLD grown ZnO-K3[Fe(CN)6] composite thin film for biosensing application

Zinc oxide-potassium ferricyanide (ZnO-KFCN) composite film was prepared on ITO coated corning glass using pulsed laser deposition (PLD). The composite film has proved to be a suitable platform for enzyme immobilization. The composite matrix exhibits the advantages of ZnO along with enhanced redox property due to the presence of a mediator in the matrix. Glucose oxidase (GOx) has been chosen as the model enzyme for studying the application of the present matrix to biosensing. The sensing response of the bio-electrode, GOx/ZnO-KFCN/ITO/glass, towards glucose was studied by electrochemical and photometric techniques. The bio-electrode exhibits good linearity and low value of Michaelis-Menten constant. Due to efficient biosensing in a mediator-less system the present bio-electrode should lead to a hand held integrated lab-on-chip device.     

Glucose fast-response amperometric sensor based on glucose oxidase immobilized in an electropolymerized poly(o-phenylenediamine) film

o-Phenylenediamine has been used for glucose oxidase (GOx) immobilization on Pt electrodes by electrochemical polymerization at +0.65 V vs SCE. By this approach the enzyme is entrapped in a strongly adherent, highly reproducible thin membrane, whose thickness is around 10 nm. This one-step procedure produces a glucose sensor with a response time less than 1 s, an active enzyme loading higher than 3 units/cm2 of electrode surface, a high sensitivity, and a sufficiently wide linear range. The glucose response shows an apparent Michaelis-Menten constant, K'm = 14.2 mM, and a limiting current density, jmax of 181 microA/cm2. The product kD of partition and diffusion coefficients of glucose in the polymer film is on the order of 10(-13) cm2/s. Due to permselectivity characteristics of the membrane, the access of ascorbate, a common interfering species, to the electrode surface is blocked. To our knowledge, this represents the first report of a membrane capable, at the same time, of immobilizing GOx and rejecting ascorbate. The interesting electrode behavior can be rationalized by using an existing model predicting the amperometric response of an immobilized GOx system.     

Immobilization of enzymes through one-pot chemical preoxidation and electropolymerization of dithiols in enzyme-containing aqueous suspensions to develop biosensors with improved performance

A protocol of one-pot chemical preoxidation and electropolymerization of monomers (CPEM) in enzyme-containing aqueous suspensions (or solutions) was proposed as a universal strategy for high-activity and high-load immobilization of enzymes to construct amperometric biosensors, which was proven to be effective for the monomer of 1,4-benzenedithiol (BDT), 1,6-hexanedithiol, o-phenylenediamine, o-aminophenol or pyrrole, the preoxidant of K3Fe(CN)6 or p-benzoquinone, and the enzyme of glucose oxidase (GOx) or alkaline phosphatase (AP) to develop GOx-based glucose biosensors or AP-based disodium phenyl phosphate biosensors. As a case examined in detail, a well-dispersed aqueous suspension of the poorly soluble BDT was obtained through its dispersion assisted by ultrasonication and coexisting GOx, which was then subject to chemical preoxidation through adding K3Fe(CN)6, yielding many composites of insoluble BDT oligomers with lots of high-activity enzyme molecules entrapped. Some insoluble composites were then electrochemically codeposited with poly(1,4-benzenedithiol) on an Au electrode, yielding an enzyme film with high-load and high-activity enzyme immobilized. The glucose biosensor prepared here from the CPEM protocol showed much better performance than that from the preoxidant-free conventional electropolymerization (CEP) protocol, with a detection sensitivity increase by a factor of 32 in this case. The GOx-based and AP-based first-generation biosensors developed from the present CPEM protocol all exhibited notably improved performance compared with the analogues from the preoxidant-free CEP protocol. The electrochemical quartz crystal microbalance (EQCM) technique was used to investigate various electrode modification processes. The values of quantity and enzymatic specific activity (ESA) of the immobilized enzymes were evaluated through the EQCM and the conventional UV-vis spectrophotometric method, given that the CPEM protocol notably improved the quantity and the ESA of immobilized enzymes as compared with the preoxidant-free CEP protocol. The proposed CPEM protocol may be interesting in a number of fields, including biosensing, biocatalysis, biofuel cells, bioaffinity chromatography, and biomaterials, and the successful electropolymerization of dithiols in aqueous suspensions (two-phase electropolymerization) may open a new avenue for many monomers that are poorly soluble in neutral aqueous solutions to in situ immobilize biomolecules for bioapplications.     

Improvement of homogeneity of analytical biodevices by gene manipulation

Homogeneity is proposed for evaluation of the quality of analytical biodevices, such as biosensors and biochips. As a demonstration, glucose oxidase (GOx) was modified at its C-terminal with a linker peptide with a cysteine residue at the end. The fusion structure (GOx-linker-cysteine) enables the enzyme to immobilize on gold surfaces with a Cys-S-Au bond or to immobilize on a silanized glass surface via disulfide chemistry. With this fusion structure, the enzyme can be anchored onto the substrate with well-controlled orientation, thus forming a homogeneous biological layer on biodevices. The linker peptide between GOx and the cysteine acts as a spacer to reduce the steric hindrance caused by the bulky body of the enzyme. Biochemistry experiments showed that this genetically modified glucose oxidase (shortened to GOxm) retained most of its catalytic characteristics, with K(m) and K(cat) similar to those of the wild-type GOx. Electrochemistry experiments showed that GOxm-modified electrode gave higher and more stable current responses than the electrode modified with GOx which has no free -SH on its surface. The coefficients of variation (used for evaluation of the interchangeability of the enzyme device from the same batch preparation) were 9.5% for the GOxm gold electrode and 20.0% for the GOx gold electrode and the GOxm oxygen electrode. The relative errors (used for evaluation of the precision of the individual enzyme device) were 2.9% for the GOxm gold electrode, 12.0% for the GOx gold electrode, and 11.2% for the GOxm oxygen electrode. Atomic force microscopy images revealed that GOxm formed a self-assembled monolayer in a hexagonal-like lattice packing arrangement on the gold surface, while GOx formed multilayer assembling or aggregated particles. The homogeneity of the protein chips, the GOxm array that was prepared through -S-S- formation, and the GOx array that was prepared through nonspecific adsorption was evaluated. The coefficients of variation, calculated with the signal level of all dots, were 5.4% for the GOxm array and 81.8% for the GOx array. All experimental results pointed to the fact that the homogeneity of the analytical biodevices could be considerably improved by using the proposed method.     

Electron transfer reactions of glucose oxidase at Au111 electrodes modified with phenothiazine derivatives

The catalytic reaction of glucose oxidase (GOx) mediated by 3-(10-phenothiazyl)propionic acid (PT-PA) and phenothiazine-labeled poly(ethylene oxide) (PT-PEO1000) that are covalently bonded to Au(111) electrodes has been investigated. The PT-PA and PT-PEO1000 are reacted with 2-aminoethanethiol (AET), followed by the formation of a self-assembled monolayer (SAM) onto the Au surface. The PT group immobilized on the SAM of AET acts as an effective mediator for the electron transfer (ET) between the electrode and the FAD center of freely diffusing GOx in solution. The ET rate constant estimated from the catalytic current using a newly derived equation is larger by 1 order of magnitude for the PT-PA-modified system (1.1 x 10(5) dm(3) mol(-1) s(-1)) than for the PT-PEO1000 system (1.4 x 10(4) dm(3) mol(-1) s(-1)). The order of the magnitude of the ET rate constant clearly contrasts with the GOx hybrid systems that we previously investigated (Anal. Chem. 2003, 75, 910-917), in which the presence of the PEO spacer enhances the ET reaction rate. The reduction in the apparent PT concentration at the electrode interface due to the high mobility of the PEO chain, leading to low efficiency in the formation of an enzyme-mediator complex, is a possible reason for the lower mediation ability of PT-PEO1000 than that of PT-PA for the ET between the FAD group and PT(+) immobilized on the electrode. Inhibition of the penetration of GOx molecules into the monolayer and of the accessibility of some part of PT groups to GOx molecules could also be reasons for the lower mediation ability of PT-PEO1000 thickly modified on the electrode.     

Exploiting metal-organic coordination polymers as highly efficient immobilization matrixes of enzymes for sensitive electrochemical biosensing

We report on the exploitation of metal-organic coordination polymers (MOCPs) as new and efficient matrixes to immobilize enzymes for amperometric biosensing of glucose or phenols. A ligand, 2,5-dimercapto-1,3,4-thiadiazole (DMcT), two metallic salts, NaAuCl(4) and Na(2)PtCl(6), and two enzymes, glucose oxidase (GOx) and tyrosinase, are used to demonstrate the novel concept. Briefly, one of the metallic salts is added into an aqueous suspension containing DMcT and one of the enzymes to trigger the metal-organic coordination reaction, and the yielded MOCPs-enzyme biocomposite (MEBC) is then cast-coated on an Au electrode for biosensing. The aqueous-phase coordination polymerization reactions of the metallic ions with DMcT are studied by visual inspection as well as some spectroscopic, microscopic, and electrochemical methods. The thus-prepared glucose and phenolic biosensors perform better in analytical performance (such as sensitivity and limit of detection) than those prepared by the conventional chemical and/or electrochemical polymerization methods and most of the reported analogous biosensors, as a result of the improved enzyme load/activity and mass-transfer efficiency after using the MOCPs materials with high adsorption/encapsulation capability and unique porous structure. For instance, the detection limit for catechol is as low as 0.2 nM here, being order(s) lower than those of most of the reported analogues. The enzyme electrode was also used to determine catachol in real samples with satisfactory results. The emerging MOCPs materials and the suggested aqueous-phase preparation strategy may find wide applications in the fields of bioanalysis, biocatalysis, and environmental monitoring.     

A bioconjugated polyglycerol dendrimer with glucose sensing properties

In this work, the biological and electrochemical properties of glucose biosensor based on polyglycerol dendrimer (PGLD) is presented. Streptokinase (SK), glucose oxidase (GOx) and phosphorylcholine (PC) were immobilized onto PGLD to obtain a blood compatible bioconjugate with glucose sensing properties. The bioconjugated PGLD was entrapped in polyaniline nanotubes (PANINT's) through template electrochemical polymerization of aniline. PANINT's were used as electron mediator due to their high ability to promote electron-transfer reactions involving GOx. Platelet adhesion, fibrinolytic activity and protein adsorption were studied by in vitro experiments to examine the interaction of blood with PGLD biosensor. The PGLD biosensor exhibits a strong and stable amperometric response to glucose. The enzyme affinity for the substrate (K (M) (app) ) indicates that the enzyme activity was not significantly altered after the bioconjugation of GOx with PGLD dendrimer. The bioelectrochemical properties suggest that the bioconjugated PGLD developed in this work appears to be a good candidate for providing interfaces for implantable biosensors, especially oxidoreductase-based sensors.     

Transforming the fabrication and biofunctionalization of gold nanoelectrode arrays into versatile electrochemical glucose biosensors

High-density arrays of conducting nanoelectrodes (i.e., nanoelectrode arrays [NEAs]) have been developed on the surface of a single electrode for numerous electrochemical sensing paradigms. However, a scalable fabrication technique and robust biofunctionalization protocol are oftentimes lacking and thus many NEA designs have limited efficacy and overall commercial viability in biosensing applications. In this report, we develop a lithography-free nanofabrication protocol to create large arrays of Au nanoelectrodes on a silicon wafer via a porous anodic alumina template. To demonstrate their effectiveness as electrochemical glucose biosensors, alkanethiol self-assembled monolayers (SAMs) are used to covalently attach the enzyme glucose oxidase to the Au NEA surface for subsequent glucose sensing. The sensitivity and linear sensing range of the biosensor is controlled by introducing higher concentrations of long-chain SAMs (11-mercaptoundecanoic acid: MUA) with short-chain SAMs (3-mercaptopropionic acid: MPA) into the enzyme immobilization scheme. This facile NEA fabrication protocol (that is well-suited for integration into electronic devices) and biosensor performance controllability (via the mixed-length enzyme-conjugated SAMs) transforms the Au NEAs into versatile glucose biosensors. Thus these Au NEAs could potentially be used in important real-word applications such as in health-care and bioenergy where biosensors with very distinct sensing capabilities are needed.     

Preparation of polypyrrole nanoparticles in reverse micelle and its application to glucose biosensor

Polypyrrole nanoparticles were successfully synthesized in cetyltrimethyl ammonium bromide (CTAB)/hexanol/water reverse micelle. The morphology and particle size of the obtained nanoparticles were characterized with transmission electron microscope (TEM) and scanning electron microscopy (SEM). Glucose biosensors were formed with glucose oxidase (GOx) immobilized in conducting composite material consisting of polypyrrole nanoparticles and ethyl cellulose. The effects of reaction conditions such as molar ratio of polypyrrole nanoparticles to ethyl cellulose, working voltage, glucose concentration, temperature and solution pH on the electrochemical response of the GOx electrode were studied. Experimental results showed that the linear range of GOx electrode was 1.0 x 10(-6)-6 x 10(-3) mol/L and the detection limit was 1.0 x 10(-7) mol/L. The electrode exhibited fine repeatability and selectability, and its lifetime was greater than one month. AFM showed that the surface of conducting composite material-glucose oxidase electrode's presents uniform granular after washing paraffin wax with cyclohexane, which was favorable for enzyme-catalyzed reaction.     

Electrochemical biosensing based on polypyrrole/titania nanotube hybrid

The glucose oxidase (GOD) modified polypyrrole/titania nanotube enzyme electrode is fabricated for electrochemical biosensing application. The titania nanotube array is grown directly on a titanium substrate through an anodic oxidation process. A thin film of polypyrrole is coated onto titania nanotube array to form polypyrrole/titania nanotube hybrid through a normal pulse voltammetry process. GOD-polypyrrole/titania nanotube enzyme electrode is prepared by the covalent immobilization of GOD onto polypyrrole/titania nanotube hybrid via the cross-linker of glutaraldehyde. The morphology and microstructure of nanotube electrodes are characterized by field emission scanning electron microscopy and Fourier transform infrared analysis. The biosensing properties of this nanotube enzyme electrode have been investigated by means of cyclic voltammetry and chronoamperometry. The hydrophilic polypyrrole/titania nanotube hybrid provides highly accessible nanochannels for GOD encapsulation, presenting good enzymatic affinity. As-formed GOD-polypyrrole/titania nanotube enzyme electrode well conducts bioelectrocatalytic oxidation of glucose, exhibiting a good biosensing performance with a high sensitivity, low detection limit and wide linear detection range.     

ZnO nanonails: synthesis and their application as glucose biosensor

Well-crystallized zinc oxide nanonails were grown in a high density by thermal evaporation process and were used as supporting matrixes for glucose oxidase (GOx) immobilization to construct efficient glucose biosensor. The GOx attached to the surfaces of ZnO nanonails had more spatial freedom in its orientation, which facilitated the direct electron transfer between the active sites of immobilized GOx and electrode surface. The fabricated biosensor showed a high sensitivity of 24.613 microA cm(-2) mM(-1) with a response time less than 10 s. Moreover, it shows a linear range from 0.1 to 7.1 mM with a correlation coefficient of R = 0.9937 and detection limit of 5 microM.     

Electrochemical behavior of gold nanoparticles modified nitrogen incorporated tetrahedral amorphous carbon and its application in glucose sensing

Gold nanoparticles (NPs) with 10-50 nm in diameter were synthesized on nitrogen incorporated tetrahedral amorphous carbon (ta-C:N) thin film electrode by electrodeposition. The deposition and nucleation processes of Au on ta-C:N surface were investigated by cyclic voltammetry and chronoamperometry. The morphology of Au NPs was characterized by scanned electron microscopy. The electrochemical properties of Au NPs modified ta-C:N (ta-C:N/Au) electrode and its ability to sense glucose were investigated by voltammetric and amperometric measurements. The potentiostatic current-time transients showed a progressive nucleation process and diffusion growth of Au on the surface of ta-C:N film according to the Scharifker-Hills model. The Au NPs acted as microelectrodes improved the electron transfer and electrocatalytic oxidation of glucose on ta-C:N electrode. The ta-C:N/Au electrode exhibited fast current response, a linear detection range of glucose from 0.5 to 25 mM and a detection limit of 120 microM, which hinted its potential application as a glucose biosensor.     

Influence of surface electrode on luminescent properties of nanocrystalline silicon electroluminescent device

We describe the electrical and luminescence properties of nanocrystalline silicon (nc-Si) based red electroluminescent (EL) devices using an indium tin oxide (ITO) and/or gold (Au) films as a surface electrode, and the variation in the transmittance and resistivity of two electrodes with various film thicknesses. The increase in the film thickness from 50 to 200 nm of the ITO electrode led to the lowering of resistivity from 2.0 x 10(-3) to 9.1 x 10(-4) omega cm and almost the same value (83-92%) of transmittance in the red region. On the other hand, the Au electrode was lowered the resistivity from 1.8 x 10(-4) to 1.6 x 10(-5) omega cm and the transmittance in the red region from 42 to 1.8% with increasing the film thickness from 10 to 80 nm. Moreover, the red luminescence from the EL devices using the ITO and/or Au electrodes having thickness of 200 and 10 nm, respectively, obtained by applying the direct current forward voltage above 4.5 and 2.5 V and/or by flowing the forward current density above 53 and 38 mA/cm2, respectively. However, the luminescence intensity of EL device with the ITO electrode strengthened more than about one order of magnitude in comparison to that of the EL device with the Au electrode. This was due to the high value of transmittance in the red region of the ITO electrode. We suggest that the ITO electrode is an optimum surface electrode for the realization of nc-Si based EL device with the high brightness.     

Integration of enzymes and electrodes: spectroscopic and electrochemical studies of chitosan-enzyme films

A new film-forming solution was developed for the efficient immobilization of enzymes on solid substrates. The solution consisted of a biopolymer, chitosan (CHIT), that was chemically modified with a permeability-controlling agent, Acetyl Yellow 9 (AY9), using glutaric dialdehyde (GDI) as a molecular tether. A model enzyme, glucose oxidase (GOx), was mixed with the CHIT-GDI-AY9 solution and cast on the surface of platinum electrodes to form robust CHIT-GDI-AY9-GOx films for glucose biosensing. UV-visible and infrared spectroscopies were used to determine the composition of the films. The optimized films contained on average 1 molecule of AY9/3 glucosamine units of chitosan and 25 free GDI tethers/1 molecule of GOx. The electrochemical assays of the films indicated both a very high efficiency of enzyme immobilization (approximately 99%) and large enzyme activity (60 units cm(-2)). The latter translated into a high sensitivity (42 mA M(-1) cm(-2)) of the Pt/CHIT-GDI-AY9-GOx biosensor toward glucose. The biosensor operated at 0.450 V, had a fast response time (t90% < or = 3 s), and was free of typical interferences, and its dynamic range covered 3 orders of magnitude of glucose concentrations. The lowest actually detectable concentration was 10 microM glucose. In addition, the biosensor displayed a practical shelf life and excellent operational stability, e.g. its response was stable during 24-h testing under continuous polarization and continuous flow of 5.0 mM glucose solution. The proposed approach to enzyme immobilization is simple, efficient, and cost-effective and should be of importance in the development of biosensors based on other enzymes that are more expensive than glucose oxidase.     

基于生物素-亲和素系统的酶固定化及纳米金增效的研究

应用石英晶体微天平(QCM)研究了基于生物素一亲和素系统的葡萄糖氧化酶的固定化,探讨了纳米金修饰QCM金基片对酶固定化的一系列过程的影响。在本实验条件下,利用生物素．亲和素系统能较好地固定化葡萄糖氧化酶,经过纳米金颗粒修饰的QCM基片对酶的吸附量比未经修饰的基片可提高1倍以上。