Electroconductive Hydrogels: Electrical and Electrochemical Properties of Polypyrrole‐Poly(HEMA) Composites

Composites of inherently conductive polypyrrole (PPy) within highly hydrophilic poly(2‐hydroxyethyl methacrylate)‐based hydrogels (p(HEMA)) have been fabricated and their electrochemical properties investigated. The electrochemical characteristics observed by cyclic voltammetry suggest less facile reduction of PPy within the composite hydrogel compared to electropolymerized PPy, as shown by the shift in the reduction peak potential from ?472 mV for electropolymerized polypyrrole to ?636 mV for the electroconductive composite gel. The network impedance magnitude for the electroconductive hydrogel remains quite low, ca. 100 Ω, even upon approach to DC, over all frequencies and at all offset potentials suggesting retained electronic (bipolaronic) conductivity within the composite. In contrast, sustained application of +0.7 V (vs. Ag/AgCl, 3 M Cl^?) for typically 100 min. (conditioning) to reduce the background amperometric current to <1.0 μA, resulted in complete loss of electroactivity. Nyquist plots suggest that sustained application of such a modest potential to the composite hydrogel results in impedance characteristics that resembles p(HEMA) without evidence of the conducting polymer component. PPy composite gels supported a larger ferrocene monocarboxylate diffusivity (D_appt=7.97×10^?5 cm² s^?1) compared to electropolymerized PPy (D_appt=5.56×10^?5 cm² s^?1), however a marked reduction in diffusivity (D_appt=1.01×10^?5 cm² s^?1) was observed with the conditioned hydrogel composite. Cyclic voltammograms in buffer containing H₂O₂ showed an absence of redox peaks for electrodes coated with PPy‐containing membranes, suggesting possible chemical oxidation of polypyrrole by the oxidant     

Synthesis and Characterization of Some N-substituted Polypyrrole Derivatives: Towards Glucose Sensing Electrodes

Synthesis and chemical oxidative polymerization of some N-substituted pyrroles ((N-Py)s) were carried out using Clauson-Kaas method and FeCl₃ in CHCl₃ medium, respectively. The produced polymers, N-(p-benzoic acid)polypyrrole (NpbPPy); N-(o-aminophenyl)polypyrrole (NoaPPy); N-(m-nitrophenyl) polypyrrole (NmnPPy), were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) analysis. An enzymatic glucose biosensor is fabricated through immobilizing glucose oxidase (GOX) into N-substitued polypyrrole matrixes. The parameters affecting the fabrication and experimental conditions of biosensors were optimized. The best biosensor results were obtained for NpbPPy matrix. The sensitivity of the proposed biosensor permitted the determination of glucose in the concentration range of 0–9.2 M with a detection limit of 1× 10^{? 6}. The apparent Michaelis–Menten constant (K_M ^app) for the sensor was found to be 25.95 mM.     

L-Dopa Synthesis on Conducting Polymers

With regards to the synthesis of L-Dopa (l-3,4-dihydroxy phenylalanine) two types of biosensors were designed by immobilizing tyrosinase on conducting polymers; polypyrrole (PPy) and poly(3,4-ethylenedioxythiophene) (PEDOT). PPy and PEDOT were synthesized electrochemically and tyrosinase immobilized by entrapment during electropolymerization. The kinetic parameters of the designed biosensors, maximum reaction rate of the enzyme (V_max) and Michaelis Menten constant (K_m) were determined. V_max were found as 0.013 for PPy matrix and 0.041 μ mol/min.electrode for PEDOT matrix. K_m values were determined as 3.7 and 5.2mM for PPy and PEDOT matrices respectively. Calibration curves for enzyme activity vs. substrate concentration were drawn for the range of 0.8 to 2.5 mM L-Tyrosine. Optimum temperature and pH, operational and shelf life stabilities of immobilized enzyme were also examined.     

Self-encapsulation of oxidases as a basic approach to tune the upper detection limit of amperometric biosensors

This study describes a new, basic procedure for the tuning of some analytical parameters of enzymatic biosensors that are based on hydrogen peroxide-producing oxido-reductases. An amperometric biosensor based on glucose oxidase (GOx) (EC 1.1.3.4) from Penicillum vitale, immobilized on a carbon rod electrode by cross-linking with glutaraldehyde, was exploited as a model system for demonstration of the approach described here. Such an important analytical parameter as the upper detection limit was dramatically changed by the formation of a polypyrrole conducting polymer layer by the GOx-induced polymerization of polypyrrole (Ppy). An increase in the upper detection limits for differently modified electrodes was estimated by calculation of the apparent Michaelis-Menten constant [K(M(app))]. A significant increase in the long-term stability of the GOx-based electrode modified by Ppy (GOx/Ppy) was detected compared with that of an unmodified one. Further application of this approach, based on the self-encapsulation of glucose oxidase and other oxidases, is predicted for such biosensors where extension of the detection rate as well as K(M(app)) are required.     

Pt-polyaniline nanocomposite on boron-doped diamond electrode for amperometic biosensor with low detection limit

Boron-doped diamond electrodes covered with a nanostructured Pt nanoparticle-polyaniline composite have been fabricated and employed as sensitive amperometric sensors with low detection limit. A highly conductive boron-doped diamond thin film (BDD) was prepared by chemical vapor deposition, and its morphology was characterized by scanning electron microscopy and transmission electron microscopy. The nanostructured composite layer was grown on the BDD electrode by electrochemical deposition of polyaniline and Pt nanoparticles. Glucose oxidase (GOx) was then adsorptively immobilized on the modified BDD electrode. The biosensor displays a large surface area, high catalytic activity of the Pt nanoparticles, efficient electron mediation through the conducting polymer, and low background current of the electrode. The biosensor exhibits an excellent response to glucose, with a broad linear range from 5.9 μM to 0.51 mM, a sensitivity of 5.5 μA·mM^?1, a correlation coefficient (R) of 0.9947, and a detection limit of 0.10 μM. The apparent Michaelis-Menten constant (K _M ^app ) and the maximum current density of the electrode are 4.1 mM and 0.021 mA, respectively. This suggests that the immobilized GOx possesses a higher affinity for glucose at the lower K _M ^app , and that the enzymatic reaction rate constitutes the rate-limiting step of the response.     

Biosensors Based on Aligned Carbon Nanotubes Coated with Inherently Conducting Polymers

The use of multiwalled aligned carbon nanotubes provides a novel electrode platform for inherently conducting polymer based biosensors. The example used here to highlight the usefulness of such a platform is the polypyrrole based glucose oxidase system for detection of glucose. The use of these three dimensional electrodes offers advantages in that large accessible enzyme loadings can be obtained within an ultrathin layer. It has also been found that the detection of H₂O₂ at these new electrode structures containing iron loaded nanotube tips can be achieved at low anodic potentials. The result is a sensitive and selective glucose sensor.     

Catalase adsorption onto Cibacron Blue F3GA and Fe(III)-derivatized poly(hydroxyethyl methacrylate) membranes and application to a continuous system

Poly(2-hydroxyethyl methacrylate) (poly(HEMA)) membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (a-a′-azobisisobutyronitrile, AIBN). An affinity dye, i.e. Cibacron Blue F3GA (CB) was incorporated covalently and then complexed with Fe(III) ions. The polyHEMA-CB and polyHEMA-CB-Fe(III) derivatized membranes were used in the adsorption of catalase (CAT). The enzyme-loading capability of the Fe(III)-containing membrane (23.6 μg/cm²) was greater than that of the poly(HEMA)-CB derivatized membrane (17.1 μg/cm²). The adsorption phenomena appeared to follow a typical Langmuir isotherm. The K_m values for both immobilized catalases (poly(HEMA)-CB-CAT (22.4 mM) and poly(HEMA)-CB-Fe(III)-CAT (19.3 mM)) were higher than that of free enzyme (16.5 mM). Optimum operational temperature was 5°C higher than that of the free enzyme and was significantly broader. A similar observation was made for the optimum pH. Operational, thermal and storage stabilities were found to increase with immobilization, especially in the presence of Fe(III). It was observed that enzyme could be repeatedly adsorbed and desorbed without significant loss in adsorption capacity or enzyme activity.     

New Amperometric Cholesterol Biosensors Using Poly(ethyleneoxide) Conducting Polymers

Accumulation of cholesterol in human blood can cause several health problems such as heart disease, coronary artery disease, arteriosclerosis, hypertension, cerebral thrombosis, etc. Therefore, simple and fast cholesterol determination in blood is clinically important. In this study, two types of amperometric cholesterol biosensors were designed by physically entrapping cholesterol oxidase in conducting polymers; thiophene capped poly(ethyleneoxide)/polypyrrole (PEO-co-PPy) and 3-methylthienyl methacrylate-co-p-vinyl benzyloxy poly(ethyleneoxide)/polypyrrole (CP-co-PPy). PEO-co-PPy and CP-co-PPy were synthesized electrochemically and cholesterol oxidase was immobilized by entrapment during electropolymerization. The amperometric responses of the enzyme electrodes were measured by monitoring oxidation current of H₂O₂ at +0.7 V in the absence of a mediator. Kinetic parameters, such as Km and Imax, operational and storage stabilities, effects of pH and temperature were determined for both entrapment supports. Km values were found as 1.47 and 5.16 mM for PEO-co-PPy and CP-co-PPy enzyme electrodes, respectively. By using these Km values, it can be observed that ChOx immobilized in PEO-co-PPy shows higher affinity towards the substrate.     

Direct Electrochemistry of Glucose Oxidase at Reduced Graphene Oxide and β‐Cyclodextrin Composite Modified Electrode and Application for Glucose Biosensing

A simple glucose biosensor has been developed based on direct electrochemistry of glucose oxidase (GOx) immobilized on the reduced graphene oxide (RGO) and β‐cyclodextrin (CD) composite. A well‐defined redox couple of GOx appears with a formal potential of ～?0.459 V at RGO/CD composite. A heterogeneous electron transfer rate constant (K_s) has been calculated for GOx at RGO/CD as 3.8 s^?1. The fabricated biosensor displays a wide response to glucose in the linear concentrations range from 50 µM to 3.0 mM. The sensitivity and limit of detection of the biosensor is estimated as 59.74 µA mM^?1 cm^?2 and 12 µM, respectively.     

Amperometric Glucose Biosensing of Gold Nanoparticles and Carbon Nanotube Multilayer Membranes

A novel multilayer gold nanoparticles/multiwalled carbon nanotubes/glucose oxidase membrane was prepared by electrostatic assembly using positively charged poly(dimethyldiallylammonium chloride) to connect them layer by layer. The modification process and membrane structures were characterized by atomic force microscopy, scanning electron microscopy and electrochemical methods. This membrane showed excellent electrocatalytic character for glucose biosensing at a relatively low potential (?0.2 V). The K_m value of the immobilized glucose oxidase was 10.6 mM. This resulting sensor could detect glucose up to 9.0 mM with a detection limit of 128 μM and showed excellent analytical performance.     

Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane

Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters K_m and V_max for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors.     

Micro- to nanostructured poly(pyrrole-nitrilotriacetic acid) films via nanosphere templates: applications to 3D enzyme attachment by affinity interactions

We report the combination of latex nanosphere lithography with electropolymerization of N-substituted pyrrole monomer bearing a nitrilotriacetic acid (NTA) moiety for the template-assisted nanostructuration of poly(pyrrole-NTA) films and their application for biomolecule immobilization. The electrodes were modified by casting latex beads (100 or 900 nm in diameter) on their surface followed by electropolymerization of the pyrrole-NTA monomer and the subsequent chelation of Cu²⁺ ions. The dissolution of the nanobeads leads then to a nanostructured polymer film with increased surface. Thanks to the versatile affinity interactions between the (NTA)Cu²⁺ complex and histidine- or biotin-tagged proteins, both tyrosinase and glucose oxidase were immobilized on the modified electrode. Nanostructuration of the polypyrrole via nanosphere lithography (NSL) using 900- and 100-nm latex beads allows an increase in surface concentration of enzymes anchored on the functionalized polypyrrole electrode. The nanostructured enzyme electrodes were characterized by fluorescence microscopy, 3D laser scanning confocal microscopy, and scanning electron microscopy. Electrochemical studies demonstrate the increase in the amount of immobilized biomolecules and associated biosensor performances when achieving NSL compared to conventional polymer formation without bead template. In addition, the decrease in nanobead diameter from 900 to 100 nm provides an enhancement in biosensor performance. Between biosensors based on films polymerized without nanobeads and with 100-nm nanobeads, maximum current density values increase from 4 to 56 μA cm^?2 and from 7 to 45 μA cm^?2 for biosensors based on tyrosinase and glucose oxidase, respectively.     

Electrochemically prepared glucose biosensors: kinetic and faradaic processes involving ascorbic acid and role of the electropolymerized film in preventing electrode-fouling

The influence is discussed of ascorbic acid (AA) on the response of a glucose biosensor based on glucose oxidase immobilized in electropolymerized poly(ophenylenediamine) (PPD) or overoxidized poly(pyrrole) (oxPPy) films on a Pt electrode. The kinetics of the homogeneous reaction between AA and H₂O₂ has been investigated by two independent methods and found to be too slow to influence the response of typical glucose biosensors. Therefore, the decrease in the sensor response, observable when working in batch under typical experimental conditions, can in no way be ascribed to a depletion of H₂O₂, which is produced in the biocatalytic cycle via the homogeneous reaction with AA. While the purely additive Faradaic interference can practically be nullified by both entrapping membranes, electrode fouling by electro-oxidation products of AA (responsible for the observed decrease in glucose sensitivity) might still represent a problem when working with PPD based biosensors. In this respect the permeability characteristics of the film markedly influence the magnitude of the observed phenomena. The oxPPy film achieves the goal of completely eliminating ascorbate without blocking the access of glucose to the immobilized enzyme. Permanent address: Dipartimento di Chimica, Universita' della Basilicata, Via N. Sauro 85, I-85100 Potenza, Italy     

Amperometric enzyme electrode for glucose determination based on poly(pyrrole-2-aminobenzoic acid)

Polymerization of pyrrole and 2-aminobenzoic acid has been investigated, and a functionalized stable film of poly(pyrrole-2-aminobenzoic acid) (PP2ABA) has been obtained electrochemically onto platinum electrode. Different cyclic voltammetric behavior is obtained for polypyrrole and PP2ABA during electrosynthesis. Fourier-transformed infrared spectrometry and surface-enhanced Raman spectrometry measurements on the two films have confirmed the presence of carboxylate group in the films. The enzyme, glucose oxidase, was covalently immobilized on a conducting PP2ABA film, and amperometric response was measured as a function of concentration of glucose at a potential of 0.7 V vs Ag/AgCl in 0.1 M phosphate buffer at pH 6.2. The effect of polymeric film thickness, pH, and possible interferents were investigated. The linear range of the calibration curve is from 3 to 40 mM with a sensitivity of 0.058 μA mM⁻¹ cm⁻² and a limit of detection of 0.5 mM. The apperent Mishaelis–Menten constant K _M is calculated to be 1 × 10⁻² mM, and the response time is 5 s.     

仿生制备有机-无机复合微囊固定化葡萄糖氧化酶

将层层自组装技术与仿生矿化技术相结合,由聚苯乙烯磺酸钠、聚二甲基二烯丙基氯化铵和二氧化硅成功制备(聚苯乙烯磺酸钠-聚二甲基二烯丙基氯化铵)₂-二氧化硅复合微囊.采用扫描电子显微镜、红外光谱和热重对微囊的形貌和化学结构进行了表征.以该复合微囊作为理想载体固定化葡萄糖氧化酶.结果表明,固定于复合微囊中的葡萄糖氧化酶的热稳定性、pH稳定性、操作稳定性得到了提高;在最适条件下,复合微囊固定化葡萄糖氧化酶的酶活回收率为72.85%,米氏常数是游离葡萄糖氧化酶的2.21倍.复合微囊在化学/生物催化、药物/基因传递系统和生物传感器应用方面具有一定的潜能.     

Minimization of Interferences in Flow Injection Amperometric Glucose Biosensor Based on Oxidation of Enzymatically‐produced H2O2

One of the major problems in amperometric biosensors based on detection of H₂O₂ produced by enzymatic reaction between oxidase enzymes and substrate is the interference of redox active compounds such as ascorbic acid (AA), dopamine (DA) and uric acid (UA). To minimize these interferences, sodium bismuthate was used for the first time as an insoluble pre‐oxidant in the flow injection (FI) amperometric glucose biosensor at a Glucose oxidase (GOx) immobilized Pt/Pd bimetallic modified pre‐anodized pencil graphite electrode (p.PGE). In this context, these interfering compounds were injected into a flow injection analysis (FIA) system using an injector which was filled with NaBiO₃. Thus, these interferents were converted into their redox inactive oxidized forms before reaching the electrode in the flow cell. While glucose was not influenced by the pre‐oxidant in the injector, the huge oxidation peak currents of the interferents decreased significantly in the biosensor. FI amperometric current time curves showed that the AA, DA and UA were minimized by 96 %, 86 %, and 98 % respectively, in the presence of an equivalent concentration of interferences in a 1.0 mM glucose solution. The proposed FI amperometric glucose biosensor exhibits a wide linear range (0.01–10 mM, R²=0.9994) with a detection limit of 2.4×10^?3 mM. Glucose levels in the artificial serum and two real samples were successfully determined using the fabricated FI amperometric biosensor.     

A New Technique for Chemical Deposition of Pt Nanoparticles and its Applications on Biosensor Design

A new glucose biosensor design based on glucose oxidase (GOD) immobilized by polypyrrole has been described in this paper. The polymerization of pyrrole was initiated by a hexachloroplatinate which itself was reduced into Pt nanoparticles and thus served as a catalyst for the H₂O₂ oxidation. Properties of the produced GOD modified electrode were examined and the activity of the entrapped enzyme was determined by basic application on the amperometric detection of glucose. Much better results were found comparatively with the enzyme electrode for which the enzyme was entrapped by the electrochemically polymerized polypyrrole. This kind of technique for Pt nanoparticles deposition can be generalized to many cases where polypyrrole is used.     

Immobilization of Urease on (HEMA/IA) Hydrogel Prepared by Gamma Radiation

In the present study, the copolymeric hydrogels based on 2-hydroxyethyl methacrylate (HEMA) and itaconic acid (IA) were synthesized by gamma radiation induced radical polymerization, in order to examine the potential use of these hydrogels in immobilization of Citrullus vulgaris urease. Gelation and Swelling properties of PHEMA and copolymeric P (HEMA/IA) hydrogels with different IA contents (96.5/3.5, 94.4/5.6 and 92.5/7.5 mol) were studied in a wide pH range. Initial studies of so-prepared hydrogels show interesting pH sensitivity in swelling and immobilization. C. vulgaris urease was immobilized on HEMA/IA (92.5/7.5) at 6 kGy with 41.3% retention of activity. The properties of free and immobilized urease were compared. Immobilized urease maintained a higher relative activity than free urease at both lower and higher pH levels, indicating that the immobilized urease was less sensitive to pH changes than the free urease. The K_m value of the immobilized urease was approximately 2 times higher than that of the free urease. Temperature stability was improved for immobilized enzyme. The free form exhibited a loss about 80% of activity upon incubation for 15 min at 80°C. The influence of various heavy metal ions at the concentration of l mM was improved after enzyme immobilization. The immobilization of C. vulgaris urease on HEMA/IA (92.5/7.5) at 6 kGy showed a residual activity of 47 % after 4 reuses.     

Composite Material Based on Macroporous Polyaniline and Osmium Redox Complex for Biosensor Development

Here the feasibility of layers based on the conducting polymer polyaniline (PANI) as component of glucose biosensors using glucose oxidase (GOx) as enzyme and [Os(bpy)₂(4‐aminomethylpyridine)Cl]PF₆ (OsCmplx) as electrochemical mediator, is evaluated. Particularly, PANI was employed to obtain a nanostructured macroporous material (m‐PANI) around polystyrene nanoparticles taken as template and the mediator was co‐immobilized during the polymerizing procedure. The GOx biosensor based on OsCmplx modified m‐PANI provides a linear response to glucose concentration in the range 5 up to 65 mM with a sensitivity of 3.54 µA/mM/cm² (on a projected geometric area=0.07 cm²), an LOD of 0.8 mM and a good precision (%RSD≤7, n=5); the biosensor is stable showing a decrease of 10% to the value of the sensitivity after 15 days of use and of about 50% after 40 days.     

Improvement of poly(amphiphilic pyrrole) enzyme electrodes via the incorporation of synthetic laponite-clay-nanoparticles

The electropolymerization of an enzyme-amphiphilic pyrrole ammonium-laponite nanoparticles mixture preadsorbed on the electrode surface provides the simultaneous immobilization of the enzyme and the hydrophilic laponite-clay-nanoparticles in a functionalized polypyrrole film. The presence of incorporated laponite particles within the electrogenerated polymer induces a strong improvement of the analytical performances (I_max and sensitivity) of amperometric biosensors based on polyphenol oxidase. These beneficial effects have been attributed to a marked enhancement of the apparent specific activity of the immobilized enzyme (from 0.21 to 0.85% of the specific activity of the free enzyme), the permeability of the host polymer being unchanged. This strategy of biosensor performance improvement was tested with cholesterol oxidase as an enzyme model. The presence of laponite additive in the poly(amphiphilic pyrrole) host matrix induces a similar enhancement of sensitivity and I_max for cholesterol biosensing as well as a large improvement of the storage stability of the polypyrrole-cholesterol oxidase electrode.