Enzyme electrodes based on organic metals

The parameters of enzyme electrodes based on organic metals are presented. Cytochrome b₂ (E.C. 1.1.2.3), glucose oxidase (E.C. 1.1.3.4), xanthine oxidase (E.C. 1.2.3.2) and peroxidase (E.C. 1.11.1.7) were used in electrodes sensitive to l-lactate, glucose, hypoxanthine and hydrogen peroxide. Electrocatalytic oxidation of NADH on organic metals and ethanol and acetaldehyde sensitive electrodes containing alcohol dehydrogenase (E.C. 1.1.1.1) were studied. Biocatalytic charge accumulation, the mechanism of electron exchange between the enzyme active centres and organic metals, and the future application of organic metals are discussed.     

Enzyme electrodes based on sono-gel containing ferrocenyl compounds

An amperometric-mediated glucose sensor has been developed by employing a silica sono-gel carbon composite electrode (SCC). The chosen mediators, ferrocene (Fc) and 1,2-diferrocenylethane (1), have been immobilized in the sono-gel composite matrix. The complex 1 has been employed for the first time as an electron transfer mediator for signal transduction from the active centre of the enzyme to the electrode conductive surface. After the optimisation of the construction procedure the best operative conditions for the analytical performance of the biosensor have been investigated in terms of pH, temperature and applied potential. Cyclic voltammetric and amperometric measurements have been used to study the response of both the glucose sensors, which exhibit a fast response and good reproducibility. The sensitivity to glucose is quite similar (6.7+/-0.1 microA/mM versus 5.3+/-0.1 microA/mM) when either Fc or 1 are used as mediators as are the detection limit ca. 1.0 mM (S/N=3) and the range of linear response (up to 13.0 mM). However, the dynamic range for glucose determination results wider when using 1 (up to 25.0 mM). The apparent Michaelis-Menten constants, calculated from the reciprocal plot under steady state conditions, are 27.7 and 31.6 mM for SCC-Fc/GOx and SCC-1/GOx electrodes, respectively, in agreement with a slightly higher electrocatalytic efficiency for the mediator 1.     

Conductometric immunosensors for the detection of staphylococcal enterotoxin B based bio-electrocalytic reaction on micro-comb electrodes

Staphylococcal enterotoxin B (SEB) is one of many toxins produced by the Gram-positive bacterium Staphylococcal aureus. While SEB is known as the causative agent of certain food poisonings it is also considered abiological select agent. Thus, rapid and accurate identification of SEB during either surveillance or in response to a biothreat is critical to the mitigation of the suspect agent. This report presents a new conductometric immune-biosensor for the detection of SEB based on immobilization of horseradish peroxidase (HRP)-labeled SEB antibody (HRP-anti-SEB) onto nanogold/chitosan-multiwalled carbon nanotube (Au/CTS-MWNT)-functionalized biorecognition interface. The formation of the antibody-antigen complex by a simple one-step immunoreaction between the immobilized HRP-anti-SEB and SEB in sample solution introduced a barrier of electrical communication between the immobilized HRP and the base surface, thus local conductivity variations could be evaluated by the bio-electrocatalytic reaction of HRP in 0.02 M PBS (pH 6.8) containing 0.15 mM H(2)O(2), 0.06 M KI and 0.1 M NaCl. Under optimal conditions, the proposed immune-biosensor exhibited a good conductometric response relative to SEB concentration in a linear range from 0.5 to 83.5 ng/ml with a correlation coefficient of 0.998. The developed immune-biosensor showed an acceptable accuracy, reproducibility and stability. Milk samples spiked with various concentrations of SEB gave an average of 116% recovery of the toxin.     

Screen-printed electrodes with electropolymerized Meldola Blue as versatile detectors in biosensors

Electropolymerization of Meldola Blue was carried out by cyclic voltammetry in the range from -0.6 to +1.4 V vs. Ag/AgCl, thus defining a new immobilization procedure of the phenoxazine mediator on screen-printed graphite electrodes. Evidence of polymer formation was provided by electrochemical and Fourier transform infrared spectroscopy (FTIR) data. Following polymerization, Meldola Blue preserved the ability to catalyze NADH oxidation allowing to achieve a detection limit of 2.5 x 10(-6) mol l(-1) and a sensitivity of 3713 microA l mol(-1) in amperometric determinations at 0 V vs. Ag/AgCl. In addition, the polymeric mediator was found to facilitate the reduction of hydrogen peroxide in the absence of peroxidase. Typical calibration at -0.1 V vs. Ag/AgCl shows a detection limit of 8.5 x 10(-5) mol l(-1), a sensitivity of 494 microA l mol(-1) and a linear range from 2.5 x 10(-4) to 5 x 10(-3) mol l(-1) hydrogen peroxide.     

Novel renewable immunosensors based on temperature-sensitive PNIPAAm bioconjugates

A novel renewable immunosensor was created comprising a temperature-controlled surface composed of poly(n-isopropylacrylamide) (PNIPAAm)-antibody conjugates that could reversibly bind the antigen. Bovine serum albumin (BSA) and the corresponding antibody (anti-BSA) were chosen as a model antibody-antigen system to demonstrate the concept. The thermally responsive PNIPAAm conjugated to anti-BSA displayed a controllable conformation change between an expanded and a collapsed form, below and above its characteristic phase transition temperature, i.e. low critical solution temperature (LCST). This showed a remarkable change in the bioaffinity of the conjugate for BSA. Thus, a renewable anti-BSA surface was generated for re-binding of the target antigen at the thermally controllable PNIPAAm-anti-BSA conjugated surface. The temperature-controlling strategy resulted in the regeneration of immunosensors on which immobilized anti-BSA antibodies retained their activity and specificity for more than 30 reproducible assays. The level of dissociation reached 89%, which is comparable with established recovery methods, while offering easer handing. The controlled binding and dissociation were monitored by quartz crystal microbalance (QCM), confocal fluorescence, native electrophoresis, laser-induced fluorescence, and electrochemical impedance methods.     

Impedimetric immunosensors based on the conjugated polymer PPV

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.     

Novel amperometric immunosensors based on iridium oxide matrices

Novel immunosensors based on antibodies immobilized in electrochemically grown iridium oxide (IrOx) thin film matrices have been developed. Antibody loading in the oxide was evaluated using a non-competitive electrochemical immunoassay for IgG. Anti-IgG loading in the oxide was found to be dependent on the concentration of anti-IgG present in the oxide growth step, with 400 microg/ml anti-IgG producing maximum amperometric responses.To study the potential analytical properties of the matrix, the dose-response behavior of the sensors was determined using optimized alkaline phosphatase-linked IgG immunoassay. Hydroquinone diphosphate (HQDP) was used as enzyme substrate and the oxidation of hydroquinone was detected amperometrically at +420 mV. The sensors displayed a linear dose-response behavior for IgG concentrations between 10 and 200 ng/ml, saturating above 600 ng/ml, and had a low detection limit of 8 ng/ml.Finally, the method was used to produce sensors containing immobilized anti-transferrin. Using a non-optimized electrochemical immunoassay for human transferrin (HT), dose-response behavior was observed for HT concentrations between 100 and 600 ng/ml.The results presented in this paper show that IrOx matrices represent a new method for immunosensor fabrication. The oxide acts as a hydrophilic, highly porous, three-dimensional matrix that can immobilize antibodies and retain their activity. The method is attractive because it offers the potential for high antibody loadings and is suitable for mass production of sensors in an easy and economical manner.     

Miniaturized pH biosensors based on electrochemically modified electrodes with biocompatible polymers

Potentiometric pH sensors based on linear polyethylenimine (L-PEI) and linear polypropylenimine(L-PPI), two synthetic enzymes and biocompatible polymers, films were prepared by electropolymerization of three different monomers: ethylenediamine (EDA), 1,3-diaminopropane (1,3-DAP) and diethylenetriamine (DETA) in order to be used in clinical, dermatological and biological applications, such as in vivo analysis. In a first step a biosensor was tested which consisted in a platinum wire protruded from glass sheath. The polymer film coated on these platinum electrodes showed good linear potentiometric responses to pH changes from pH 3 to 10. Resulting electrodes present both good reversibility and good stability versus time. The effect of the different polymer film thicknesses to potentiometric responses was also studied. This study allowed us to develop a miniaturized pH biosensor in the second step. This sensor was fabricated using photo-lithography, followed by sputtering and lift-off processes, and it included an electronic detection system. We have also successfully studied the potentiometric responses to pH changes of this device over a period of 1 month, and so we propose this new pH micro-biosensor as an alternative to classical pH sensors currently used in dermatology.     

Label-free electrochemical immunosensors based on surface-initiated atom radical polymerization

A novel label-free immunosensing strategy for sensitive detection of tumor necrosis factor-alpha antigen (TNF-α) via surface-initiated atom transfer radical polymerization (SI-ATRP) was proposed. In this strategy, the Au electrode was first modified by consecutive SI-ATRP of ferrocenylmethyl methacrylate (FMMA) and glycidyl methacrylate (GMA), and TNF-α antibody was coupled to the copolymer segment of GMA (PGMA) by aqueous carbodiimide coupling reaction. Subsequently, the target TNF-α antigen was captured onto the Au electrode surface through immunoreaction. The whole process was confirmed by scanning electron microscopy (SEM) and surface plasmon resonance (SPR) measurements. With introduction of redox polymer segment of FMMA (PFMMA) as electron-transfer mediator, the antigen-coupled Au electrode exhibited well electrochemical behavior, as revealed by cyclic voltammetry measurement. This provided a sensing platform for sensitive detection of TNF-α with a low detection limit of 3.9pgmL(-1). Furthermore, the "living" characteristics of the ATRP process can not only be readily controlled but also allow further surface functionalization of the electrodes, thus the proposed method presented a way for label-free and flexible detection of biomolecules.     

Enzyme electrodes and their application

Starting from the state of the art, principles for improving the analytical characteristics of enzyme electrodes are discussed. Coupling of appropriate amperometric electrode processes with enzyme systems, e.g. urease or aminopeptidases, results in a simplification of operation. Optimal sample frequencies are realized on the basis of enzyme membranes, with both a small characteristic diffusion time and a high enzyme activity, applied in a well-designed sample-processing system. Coupled enzyme reactions of the sequence or competition type are successfully used for extension to new analytes, e.g. inhibitors, cofactors or alternative substrates. Cyclization of the analyte enhances the sensitivity of enzyme electrodes to the nanomolar concentration range. Enzymic anti-interference layers are a tool for improving the sensor specificity. The operational characteristics of enzyme electrodes are thus adaptable to any given analytical problem.     

Urea potentiometric biosensor based on modified electrodes with urease immobilized on polyethylenimine films

The development of a new electrochemical sensor consisting in a glass-sealed metal microelectrode coated by a polyethylenimine film is described. The use of polymers as the entrapping matrix for enzymes fulfils all the requirements expected for these materials without damaging the biological material. Since enzyme immobilization plays a fundamental role in the performance characteristics of enzymatic biosensors, we have tested four different protocols for enzyme immobilization to determine the most reliable one. Thus the characteristics of the potentiometric biosensors assembled were studied and compared and it appeared that the immobilization method leading to the most efficient biosensors was the one consisting in a physical adsorption followed by reticulation with dilute aqueous glutaraldehyde solutions. Indeed, the glutaraldehyde immobilized urease sensor provides many advantages, compared to the other types of sensors, since this type of urea biosensor exhibits short response times (15–30 s), sigmoidal responses for the urea concentration working range from 1×10^−2.5 to 1×10^−1.5 M and a lifetime of 4 weeks.     

Amperometric glucose biosensors based on Prussian Blue- and polyaniline-glucose oxidase modified electrodes.

The properties of glucose sensors fabricated by immobilization of glucose oxidase in a layer of electrochemically deposited polyaniline were investigated. Selective amperometric glucose sensors were prepared by immobilization of glucose oxidase on a Prussian Blue-modified platinum electrode in a layer of polyaniline during a one-step electropolymerization procedure from phosphate buffer. The influence of ascorbic acid and acetaminophen was completely eliminated due to impermeability of polyaniline to these substances.     

Sensor and biosensor based on Prussian Blue modified gold and platinum screen printed electrodes

Gold (Au) and platinum (Pt) screen-printed electrodes were modified with Prussian Blue (PB) for the development of amperometric sensors selective for hydrogen peroxide detection. The sensors exhibited sensitivities towards H(2)O(2) equal to 2 A M(-1) cm(-2) for Au and 1 A M(-1) cm(-2) for Pt electrodes. The sensors were also employed as the basis for construction of glucose biosensors through further modification with crystallised glucose oxidase immobilised in a Nafion membrane. In order to improve the operational stability of the modified electrodes a buffer solution containing tetrabutylammonium toluene-4-sulfonate was used. The long-term performance of the sensors and biosensors were evaluated by continuous monitoring of hydrogen peroxide and glucose solutions (50 microM and 1 mM, respectively) in the flow-injection mode for 10 h.     

Glucose microbiosensor based on alumina sol-gel matrix/electropolymerized composite membrane

A procedure is described that provides co-immobilization of enzyme and bovine serum albumin (BSA) within an alumina sol-gel matrix and a polyphenol layer permselective for endogenous electroactive species. BSA has first been employed for the immobilization of glucose oxidase (GOx) on a Pt electrode in a sol-gel to produce a uniform, thin and compact film with enhanced enzyme activity. Electropolymerization of phenol was then employed to form an anti-interference and protective polyphenol film within the enzyme layer. In addition, a stability-reinforcing membrane derived from (3-aminopropyl)-trimethoxysilane was constructed by electrochemically-assisted crosslinking. This hybrid film outside the enzyme layer contributed both to the improved stability and to permselectivity. The resulting glucose sensor was characterized by a short response time (<10 s), high sensitivity (10.4 nA/mM mm(2)), low interference from endogenous electroactive species, and a working lifetime of at least 60 days.     

Reagentless detection of DNA sequences on chemically modified electrodes

Microarray analysis requires complex optical instruments and numerous reagents. Several new electrochemical methods for creating sequence-selective microlocations are emerging; such approaches potentially facilitate electrochemical (electronic) readouts of microarrays. These developments parallel the migration of glucose sensing in diabetes monitoring from optical to electrochemical methods. Recent work provides a strategy that minimizes the use of added reagents and potentially produces a reusable sensor that can be applied to continuous monitoring applications.     

A micro-potentiometric hemoglobin immunosensor based on electropolymerized polypyrrole–gold nanoparticles composite

We report a novel micro-potentiometric hemoglobin (Hb) immunosensor based on electrochemically synthesized polypyrrole (PPy)–gold nanoparticles (AuNPs) composite. PPy–AuNPs film with AuNPs uniformly distributed in it was deposited on gold electrode surface by a simple and direct procedure, without the addition of any nanoparticles or reducing agent. And this generic method makes it possible to deposite different polymers on miniaturized electrodes. With the existence of AuNPs, the antibody immobilization onto the electrode surface was facilitated. Morphology study by field emission scanning electron microscope (FE-SEM) confirms the presence of AuNPs in PPy. Based on an ion-sensitive field-effect transistors (ISFETs) integrated chip, a micro-potentiometric immunosensor for Hb and hemoglobin-A1c (HbA1c) has been constructed. The sensor response was linear over the concentration range 60–180 μg/ml Hb and 4–18 μg/ml HbA1c. The Hb concentration in whole blood samples has also been analysed, with a linear dose–response behavior between 125 and 197 μg/ml and a sensitivity of 0.20 mV μg⁻¹ ml. The measuring ranges of the developed Hb and HbA1c immunosensors meet the clinical demand for measuring the HbA1c/Hb ratio of 5–20%. This sensor results in simple and rapid differential measurement of Hb and HbA1c, and has great potential to become an inexpensive and portable device for monitoring of diabetes.     

Bioelectrocatalysis by redox enzymes at modified electrodes

Self-assembled monolayers of thiolated compounds are used as promoters for protein-electrode reactions. They provide an anchor group based on thiol chemisorptions and also a functional group for effective interaction with the protein. These interactions are often governed by electrostatic attraction. For example, for positively charged proteins, such as cytochrome c and the selenoprotein glutathione peroxidase, mercaptoalkanoic acids have been used. Clay modification of the electrode surface has been found to facilitate the heterogeneous electron transfer process for heme proteins, e.g. cytochrome c, cytochrome P450 and myoglobin. Interestingly, nucleic acids at carbon electrodes and thiol-modified double stranded oligonucleotides act as promoters of the redox communication to proteins, whereas the mechanism is still subject to controversy interpretations. By interacting the protein immobilised at the electrode with species in solution, signal chains have been constructed. The interaction can result in a simple co-ordination or redox reaction, depending on the nature of the reaction partners. For analytical purposes, e.g. biosensors, the electrochemical redox conversion of the immobilised protein is evaluated.     

Multienzyme microbiosensor based on electropolymerized o-phenylenediamine for simultaneous in vitro determination of acetylcholine and choline

The electrochemical biosensors based on poly(o-phenylenediamine) (PoPD) and acetylcholinesterase (AChE) and choline oxidase (ChO) enzymes were fabricated on carbon fibre (CF) substrate. The electropolymerized PoPD was used to reduce the interfering substances. The electrode assembly was completed by depositing functionalized carbon nano tubes (FCNTs) and Nafion (Naf). Amperometric detection of acetylcholine (ACh) and choline (Ch) were realized at an applied potential of +750 mV vs Ag/AgCl (saturated KCl). At pH 7.4, the final assembly, Naf-FCNTs/AChE-ChO((10:1))/PoPD/CF(Elip), was observed to have high sensitivity towards Ch (6.3±0.3 μA mM(-1)) and ACh (5.8±0.3 μA mM(-1)), linear range for Ch (K(M)=0.52±0.03 mM) and ACh (K(M)=0.59±0.07 mM), and for Ch the highest ascorbic acid blocking capacity (97.2±2 1mM AA). It had a response time of <5s and with 0.045 μM limit of detection. Studies on different ratio (ACh/Ch) revealed that 10:1, gave best overall response.     

Urease sensors based on differential antimony electrodes

Urea-sensitive sensors, based on immobilized urease and the differential antimony electrode, have been constructed. The calibration curve of the sensor is linear up to 1·6–2·0 mmol litre⁻¹ in the stationary or kinetic mode of operation. The sensors retain 50% of their sensitivity for 16–17 days. The analyser for urea, constructed on the basis of the sensor, ensures quick and precise urea determination in pure blood.