Thin-film biosensor for the measurement of glucose concentration in human serum and urine

Solid-state technology and pulse electroplating were used to fabricate a glucose biosensor based on hydrogen peroxide detection. This glucose biosensor was composed of thin-film electrodes, and enzyme-immobilized and deactivated enzyme-immobilized membranes. The electrodes were fabricated by metallic film deposition. Cr and Ni adhesive layers were applied successively by vapour deposition on the thermally oxidized SiO₂ isolating layer on a silicon substrate, and then the two metallic layers were patterned by the photolithographic method. Subsequently, a 1 μm thick Au layer was applied by means of pulse electroplating, forming two anodes and one common cathode in each sensor chip. On one anode, glucose oxidase (GOD) was immobilized by cross-linking with bovin serum albumin and glutaraldehyde. A deactivated GOD-immobilized membrane was formed on the other anode, which worked as a reference working electrode. A novel differential measurement system was used to treat the output signals of the two anodes by adjusting the initial position of the response curves, compensating amplifications of the individual I—V converters and treating the output signals with a subtraction circuit in order to decrease measurement error. The test results showed that the signal of ascorbic acid up to 4.5 mmol 1⁻¹ or uric acid up to 1.2 mmol 1⁻¹ was successfully cancelled. Glucose concentrations in the range 0.02–4.0 mmol/1 could be detected and an excellent linear response was obtained in the low concentration range. The correlation coefficient between the result of the enzyme electrode and the clinically enzymatic method for glucose measurement in human serum was 0.9912. Correlated results between the biosensor method and the routine clinical method for the measurement of glucose concentration in urine were obtained. The lifetime of the enzyme electrode was over 2 months.     

A Micro Planar Ampehometric Glucose Sensor Using an Isfet as a Reference Electrode

Abstract

A very small glucose sensor has been realized, which consists of a gold working electrode with a glucose oxidase immobilized membrane on it, and a gold counter electrode, all made on a sapphire substrate. By using the pH sensitive ISFET as a reference electrode, the potential for a solution, whose pH is constant, can be measured and irreversible metal electrodes, such as gold or platinum, can be used as working electrode and counter electrode. The sensor is very suitable for miniaturizing and mass production, because the Integrated Circuit (IC) fabrication process can be applied. The glucose oxidase immobilized membrane was also deposited by a lift off method, one of the IC processes. A glucose concentration, from 1 to 100 mg/dl, was measured with good linear current output.     

Microfabricated glucose biosensor with glucose oxidase entrapped in sol-gel matrix

A microfabricated glucose biosensor based on an amperometeric hydrogen peroxide electrode has been developed. A sol-gel layer with 5 A pore size and 2 mum thickness was used as the glucose oxidase entrapping matrix. The sol-gel matrix formed over the silicon-based sensor has good mechanical and chemical stability, and the ability to entrap a large amount of enzyme. The miniaturized electrode sensing system is composed of platinum as both working and counter electrodes and silver as a reference electrode. Nafion(R) coating was applied as the interference limiting layer. A series of technologies, such as standard photolithography, electron beam evaporation and image reverse lift-off were utilized for mass production allowing 143 electrodes to be produced at the same time. The effect of oxidable interferences was <10% of the background value of the sensor response. Calibration tests of a series of individual sensors manufactured from the same silicon wafer and dip coated in the same conditions, showed a highly reproducible response characteristics (linear range up to 500 mg dl(-1) and mean sensitivity of 0.54+/-0.14 nA mg(-1) dl(-1) (n=10)).     

Development of microbiosensors

Semiconductor fabrication technology was used for development of ion sensitive field effect transistor (ISFET) and micro-electrodes which have been utilized as transducers of enzyme-based microbiosensors. A urea sensor consisted of two ISFETs; one ISFET is urease-coated ISFET and the other ISFET is reference ISFET. A linear relationship was obtained between the initial rate of voltage change and the logarithm of urea concentration over the range 1.3 to 16.7 mM. ATP and hypoxanthine sensors were also developed utilizing ISFET as a transducer. Furthermore, microelectrodes such as hydrogen peroxide and oxygen sensors were prepared by the silicone fabrication technology. A glucose sensor consisted of a hydrogen peroxide electrode and immobilized glucose oxidase membrane. A linear relationship was observed between the current increase and the concentration of glucose (1–100 mg dl⁻¹). A microoxygen electrode was constructed from Au electrodes, polymer matrix containing alkaline electrolyte and a photocross-linkable polymer membrane. This electrode was used as a transducer in microglucose sensor. A microglutamic acid sensor is also described.     

The electrochemical behaviour of ferrocene in a photocurable poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) film for a glucose biosensor

A single-step fabrication of a glucose biosensor with simultaneous immobilization of both ferrocene mediator and glucose oxidase in a photocurable methacrylic film consisting of poly(methyl methacrylate-co-2-hydroxylethyl methacrylate) was reported. The entrapped ferrocene showed reversible redox behaviour in the photocured film and no significant leaching of both entrapped ferrocene and enzyme glucose oxidase was observed because of the low water absorption properties of the co-polymer films. From electrochemical studies, ferrocene entrapped in the co-polymer film demonstrated slow diffusion properties. A linear glucose response range of 2-11 mM was obtained at low applied potential of +0.25 V. The glucose biosensor fabricated by this photocuring method yielded sensor reproducibility and repeatability with relative standard deviation of <10% and long-term stability of up to 14 days. The main advantage of the use of photocurable procedure is that biosensor membrane fabrication can be performed in a single step without any lengthy chemical immobilization of enzyme.     

Preparation and analytical testing of mediator containing photolithographically patterned enzyme membrane electrodes

The application of mediators for measurements with amperometric enzyme sensors have been investigated to improve the behavior of sensors with respect to interfering substances or for working under anaerobic conditions. The aim of this investigation is to develop photolithographically patterned enzyme membranes containing mediators, which facilitate the inexpensive technological preparation of patterned sensors. Thin layer platinum electrodes were coated with the enzyme membranes and cross-linked by means of UV light. Measurements were made in a wall-jet configuration using flow injection techniques with or without oxygen in the solutions. Optimum properties can be obtained with glucose oxidase containing membranes using tetrathiafulvalenes. The interfering substances ascorbic acid, uric acid and acetaminophenol showed no influence on the glucose measurements in the range of physiological concentrations. The membrane served as a diffusion barrier; a decrease in the applied potential to 300 mV vs. SCE also improved the ratio of the glucose response to the interference response.     

Application of heated electrodes operating in a non-isothermal mode for interference elimination with amperometric biosensors

Heated electrodes were applied for the non-isothermal operation of amperometric glucose biosensors based on glucose oxidase immobilised on the electrode surface by entrapment within a polymer layer. The localised deposition of the polymer film under simultaneous entrapment of the enzyme was achieved by an electrochemically induced pH-modulation in the diffusion zone in front of the electrode, thus altering the solubility of the polymer chains. This non-manual sensor preparation protocol could be successfully used for the modification of a novel indirectly heated electrode. The non-isothermal operating mode allows working at the optimum temperature of the enzyme sensors without any thermal distortion of the bulk solution. Increased surface temperature of the sensor thus accelerates transport as well as kinetic processes, resulting in an enhanced amperometric signal.In the presence of interfering compounds such as ascorbic acid, the proposed technique allows use of the diverging thermal impact on the sensing process, for different electrochemically active compounds, for a deconvolution of the amperometric signal at different electrode temperatures. A calculation method for determination of glucose in the presence of one interfering compound is presented as a basis for a calculative interference elimination.     

A regenerable pseudo-reagentless glucose biosensor based on Nafion polymer and l,1''-dimethylferricinium mediator

Glucose oxidase was immobilized onto electrodes by co-deposition from an aqueous solution containing the diluted ion-exchange polymer Nafion. The cationic exchange property of the polymer was used to provide high local concentrations of l,1'-dimethylferricinium (DMFc⁺) mediator in the film by exchange from solution. The mediated electrodes were operated at +200 mV (vs. ), and the Nafion film was shown to reduce interfering current from ascorbate anion. Cyclic voltammetric analysis revealed a fourteen-fold increase in the effective DMFc⁺ activity at the electrode after extraction into the film. The sensitivity to glucose was 52 μA/cm²/mM in a solution containing 0.09 mM DMFc⁺, which is at least three-fold greater than reported for similar electrodes using hydrogen peroxide detection at +650 mV, with a response time of less than 1 min for a 10 μm thick membrane. Oxygen interference was significant, requiring deaeration of the solution before analysis. The electrodes exhibited no significant decrease in sensitivity for more than 50 days on storage in acetate buffer. Electrodes covered with 8000 MWCO dialysis membrane slowed the exchange of DMFc⁺ with the solution such that the Nafion film functioned as a mediator reservoir. This permitted reagentless analysis of glucose, typically capable of twenty assays when measuring concentrations between 0.1 and 1 mM. The sensitivity for glucose was 7.85 μA/cm²/mM, which is 15% of the sensitivity for the electrode without the dialysis membrane. The detection limit was 20 μM, with a linear range extending to about 3 mM, giving a dynamic range of over two orders of magnitude. Thus where some sacrifice of sensitivity and response rate may be made, the dialysis membrane cover enables multiple analyses in a reagentless biosensor scheme.     

Enzyme electrodes using ultrafiltration membranes as enzyme carriers

Two kinds of commercially available ultrafiltration membranes were used for binding enzymes. These enzyme-bound membranes were connected with oxygen electrodes to build up enzyme electrodes. Quantitative assays of glucose, sucrose, and hydrogen peroxide were carried out using these enzyme electrodes in concentration ranges for glucose from 1 to 20 mg/dl, sucrose from 0.5 to 5 mg/ml, and hydrogen peroxide from 5 to 200 ppm. Similarly, D-alanine was measured in the range 0.005 to 0.05 mg/ml, but the calibration curve was not a straight line.     

Association behavior of a nitrilotriacetic-acid-modified dye in a poly(vinyl alcohol) film containing Ni(II)-adsorbed gold nanoparticles.

The association behavior of the dyes 5(6)-carboxyfluorescein and nitrilotriacetic acid (NTA)-modified 5(6)-carboxyfluorescein (F-NTA) in a poly(vinyl alcohol) (PVA) film and in a PVA film containing metal nanoparticles is investigated. Well-dispersed gold nanoparticles (AuNps) and Ni(II)-adsorbed AuNps are formed in the PVA film using in situ photochemical fabrication method. 5(6)-carboxyfluorescein and F-NTA are doped into the films. The F-NTA forms an H-aggregate in the PVA film containing Ni(II)-adsorbed AuNps. It is suggested that the interaction between NTA and Ni(II) adsorbed on the AuNps promotes the formation of the H-aggregate.     

Mediated glucose enzyme electrodes by cross-linking films of osmium redox complexes and glucose oxidase on electrodes

Here, we report on a novel, versatile approach for the preparation of mediated enzyme electrodes, demonstrated using cross-linked films of glucose oxidase and a range of functionalised osmium complexes on graphite electrodes. Response of enzyme electrodes are optimised by evaluation of glucose response as a function of variation in ratios of [Os(2,2′-bipyridine)₂(4-aminomethyl pyridine)Cl]⁺ redox mediator, polyallylamine support and glucose oxidase enzyme cross-linked using a di-epoxide reagent in films on graphite. Lowering of the redox potential required to mediate glucose oxidation is achieved by synthesis of complexes using (4,4′-dimethyl-2,2′-bipyridine) or (4,4′-dimethoxy-2,2′-bipyridine) as a ligand instead of (2,2′-bipyridine). Enzyme electrodes prepared using the complexes based on dimethoxy- or dimethyl-substituted bipyridines provide glucose oxidation current densities of 30 and 70 μA?cm^?2 at 0.2 and 0.35 V applied potential compared to 120 μA?cm^?2 at 0.45 V for the initial enzyme electrode, under pseudo-physiological conditions in 5 mM glucose, with stability of signals proving inadequate for long-term operation. Current output and stability may be improved by selection of alternate anchoring and cross-linking methodology, to provide enzyme electrodes capable for application to long-term glucose biosensors and anodes in enzymatic fuel cells.

A study of Nafion-coated bismuth-film electrodes for the determination of trace metals by anodic stripping voltammetry

This work reports on the fabrication, characterization and applications of Nafion-coated bismuth-film electrodes (NCBFE's) for the determination of trace metals by anodic stripping voltammetry (ASV). A NCBFE was typically prepared by first applying a 5 microl drop of a 1% Nafion solution onto the surface of a glassy-carbon rotating-disk electrode. After evaporation of the solvent, the Bi film was plated on the electrode in situ(i.e. by spiking the sample with 1000 microg l(-1) of Bi(iii) and simultaneous electrolytic deposition of the metal ions and bismuth film on the electrode surface at -1.4 V) or ex-situ(i.e. by electrolytic deposition of the bismuth film in a separate solution containing 1000 microg l(-1) of Bi(iii), followed by the ASV measurement step in the sample solution). Various fabrication and operational parameters were thoroughly investigated and discussed in terms of their effect on the ASV signals. It was found that this voltammetric sensor was suitable for the determination of metals at trace levels by square-wave ASV (SWASV) due to its multi-element detection potential, improved analytical sensitivity, high resistance to surfactants, low cost, ease of fabrication, robustness, speed of analysis and low toxicity (as compared to traditional mercury electrodes). In the presence of 4 mg l(-1) of Triton X-100, the NCBFE afforded a 10-fold peak height enhancement for the Pb peak and a 14-fold enhancement for the Cd peak over a bare BFE while the determination of Zn was feasible only on the NCBFE. The limits of detection (at a signal-to-noise ratio of 3) were 0.1 microg l(-1) for Cd and Pb and 0.4 microg l(-1) for Zn for a deposition time of 10 min. Finally, the electrode was applied to different real samples (tap-water, urine and wine) for the analysis of trace metals with satisfactory results.     

A microfluidic glucose sensor incorporating a novel thread‐based electrode system

An electrochemical sensor for the detection of glucose using thread‐based electrodes and fabric is described. This device is relatively simple to fabricate and can be used for multiple readings after washing with ethanol. The fabrication of the chip consisted of two steps. First, three thread‐based electrodes (reference, working, and counter) were fabricated by painting pieces of nylon thread with either layered silver ink and carbon ink or silver/silver chloride ink. The threads were then woven into a fabric chip with a beeswax barrier molded around the edges in order to prevent leaks from the tested solutions. A thread‐based working electrode consisting of one layer of silver underneath two layers of carbon was selected to fabricate the final sensor system. Using the chip, a PBS solution containing glucose oxidase (GOx) (10 mg/mL), potassium ferricyanide (K₃[Fe(CN)₆]) (10 mg/mL) as mediator, and different concentrations of glucose (0‐25 mM), was measured by cyclic voltammetry (CV). It was found that the current output from the oxidation of glucose was proportional to the glucose concentrations. This thread‐based electrode system is a viable sensor platform for detecting glucose in the physiological range.     

Bi-functionalization of a patterned Prussian blue array for amperometric measurement of glucose via two integrated detection schemes

A novel amperometric sensor that integrates two independent measurement schemes into a single chip for detection of glucose is fabricated. The sensor uses micro-patterned Prussian blue (PB) and ferrocene modified glucose oxidase covered by a thin Nafion membrane. We have developed an amperometric sensor for the detection of glucose that integrates two measurement schemes into a single chip. For fabrication of the sensing interface, micro-contact printing was used to transfer a self-assembled monolayer template onto a gold substrate, allowing selective electrochemical deposition of a PB array. The protective layer of the PB array was subsequently removed and replaced with a layer of redox-functionalized glucose oxidase (GOx), while the entire surface was finally covered with a perm-selective GOx-Nafion membrane. A variety of surface analytical techniques, including atomic force microscopy, surface plasmon resonance imaging and spectroscopic ellipsometry were employed to characterize the composite PB array electrode. The hybrid sensing interface allowed amperometric measurements of glucose to be carried out with two independent schemes at different potentials. The cathodic current was obtained with the PB array functioning as the electrocatalyst, while the anodic current was measured at a higher potential via a mediation mechanism using the ferrocene-modified GOx. For the quantitative detection of glucose, flow-injection analysis was used, and both the operating conditions and the design parameters were optimized. Linear responses were obtained for both anodic and cathodic signals over a concentration range from 0.1 to 50 mM, with a detection limit of 75 microM. The specificity of the sensor was demonstrated with respect to ascorbic and lactic acid. The implementation of integrated detection mechanisms allows the independent measurement of amperometric signals at two separate potentials. This improves the information gathering and opens up new avenues for developing novel methods that potentially eliminate false signal readings.     

Amperometric biosensors employing an insoluble oxidant as an interference-removing agent

A strong oxidant membrane is introduced to amperometric biosensors in order to solve the problem associated with interference from readily oxidizable species. The proposed biosensors are in planar format, and are composed of four components, i.e. a base amperometric transducer, an enzyme layer, a protecting membrane, and an oxidant membrane. In this sensor format, interfering species are removed by an oxidation reaction during their diffusion through the oxidant membrane. The oxidant membrane is introduced by dispensing a mixture of an oxidant and a polymer matrix as dissolved in an organic solvent, and thus, could be easily adapted to mass fabrication of miniature biosensors. In this work, several different reagents are examined as oxidants: BaO₂, CeO₂, MnO₂ and PbO₂. Of these, PbO₂ is shown to yield biosensors with the best performance, in terms of reducing interfering signals. Two different matrix systems are devised for use in formulating oxidant membranes: hydrophilic polyurethane (HPU) and cellulose acetate incorporating poly(ethylene glycol) (CA/PEG). While the CA/PEG-type sensor displays better sensitivity and faster response behavior, the HPU-type is shown to exhibit more pronounced interference-removing ability. The analytical utility of the proposed oxidant membrane is demonstrated by fabricating amperometric glucose and creatinine sensors as the model biosensor systems, and by investigating their response characteristics.     

One-step electropolymeric co-immobilization of glucose oxidase and heparin for amperometric biosensing of glucose

Electropolymerization was used for the co-deposition of glucose oxidase and heparin onto metal electrode transducers. Such electropolymeric co-entrapment within a non-conducting poly(1,2-phenylenediamine) (PPD) film imparts both biocatalytic and anticoagulation activities onto the transducer, and greatly improves the performance of the sensor after exposure to whole blood. Essentially identical glucose signals are observed before and after exposure to blood samples. Scanning electron micrographs after such exposure reveal no platelet deposition or formation of a fibrin "clot". The effect of the heparin co-immobilization on the glucose response is examined. Improved biocompatibility is reported also in connection with a needle-type carbon paste biosensor configuration. The simultaneous localization of the enzyme and heparin offers great promise for simplifying the preparation of enzyme electrodes and designing biocompatible implantable glucose biosensors.     

Bismuth Film Voltammetric Sensor on Pyrolyzed Photoresist/Alumina Support for Determination of Heavy Metals

Voltammetric sensors based on bismuth film electrodes are an attractive alternative to other sensors for application in electroanalysis of heavy metals. Bismuth film electrodes can be formed by a similar method on the same substrates as mercury. These systems were used most frequently for simultaneous determination of heavy metals such as Pb, Cd and Zn by anodic stripping voltammetry. Our voltammetric sensor was fabricated on an alumina substrate. A photoresist film prepared by pyrolysis of positive photoresist S‐1813 SP15 on the alumina substrate was used as an electrode support for bismuth film deposition. The influence of the Nafion membrane on the measurement sensitivity of the sensor and mechanical stability of the bismuth film were investigated. The sensor was successfully applied for determination of Pb, Cd and Zn in an aqueous solution in the concentration range of 0.2 to 10 µg L^?1 by square wave anodic stripping voltammetry on an in‐situ formed bismuth film electrode with Nafion‐coating. Parameters of the sensor such as sensitivity, linearity, detection limit, repeatability and life‐time were evaluated. In the best case, the detection limits were estimated as 0.07, 0.11 and 0.63 µg L^?1 for Pb, Cd and Zn, respectively. Finally, the applicability of the sensor was tested in analysis of Pb, Cd and Zn in real samples of tap and river water using the method of standard additions.     

Porous and electrically conductive polypyrrole-poly(vinyl alcohol) composite and its applications as a biomaterial

Bulk modification of polypyrrole (PPY) with poly(vinyl alcohol) (PVA) was carried out by the electropolymerization of pyrrole in the presence of PVA in the reaction solution, with tetraethylammonium perchlorate (TEAP) as the electrolyte. The surface morphology of the as-synthesized PPY-TEAP-PVA film was investigated using scanning electron microscopy, and the film was further characterized using X-ray photoelectron spectroscopy, electrical conductivity, the water contact angle, and BET surface area measurements. The PPY-TEAP-PVA composite is electrically conductive, hydrophilic, and microporous with a high surface area. Its potential as a biomaterial was investigated with respect to its blood compatibility and function as a substrate for biosensor fabrication and cell culture. The presence of PVA in the film attenuates blood protein adsorption, and the porous nature of the PPY-TEAP-PVA film results in a 10-fold increase in the amount of glucose oxidase covalently immobilized on the film over that on a nonporous PPY film. PC12 cell attachment and growth on the PPY-TEAP-PVA film was also shown to be enhanced compared with that on tissue culture polystyrene. The attached cells proliferated and formed a monolayer on the film surface after 48 h of seeding.     

A comparative physical study of two different hydrophilic synthetic latex matrices for the construction of a glucose biosensor

Two different biodegradable latex polymers functionalised by hydroxy (1) or gluconamide (2) groups proved to be good immobilisation matrixes for glucose oxidase. The responses of these biosensors to glucose additions were measured by potentiostating the modified electrodes at 0.6 V/SCE in order to oxidise the hydrogen peroxide generated by the enzymatic oxidation of glucose in the presence of oxygen. The response of such electrodes was evaluated as a function of film thickness, pH and temperature. Rotating disk electrode experiments showed the influence of the enzyme on the structure of both latex films, namely a marked improvement in matrix permeability. The high permeability of the latex 1 based enzyme sensor (bilayer, P(m)=8.10x10(-4) cm s(-1)) resulted in a high dynamic range. Furthermore, the activation energy for a latex 1 sensor was determined to be 44.55 and 18.03 kJ mol(-1), respectively depending on the conformation of the enzyme.     

Integration of microfabricated needle-type glucose sensor devices with a novel thin-film Ag/AgCl electrode and plasma-polymerized thin film: mass production techniques

We developed an integrated array of needle-type biosensors employing a novel process of fabrication, comprising conventional semiconductor fabrication and micromachining technology. Amperometric sensing electrodes with plasma-polymerized films and a thin-film Ag/AgCl reference electrode were directly integrated on a glass substrate with thin-film process, e.g., sputtering. An enzyme was immobilized on the electrode via the plasma-polymerized film, which was deposited directly on the substrate using a dry process. The novel thin-film Ag/AgCl reference electrode showed stable potentials in concentrated chloride solutions for a long period. The plasma-polymerized film is considered to play an important role as an interfacial design between the sensing electrode and the immobilized enzyme considering that the film is extremely thin, adheres well to the substrate (electrode) and has a highly cross-linked network structure and functional groups, such as amino groups. The results showed increments of the sensor signal, probably because the plasma-polymerized film allowed a large amount of enzyme to be immobilized. The greatest advantage is that the process can permit the mass production of high-quality biosensors at a low cost.