Amperometric protein sensor - fabricated as a polypyrrole, poly-aminophenylboronic acid bilayer

An approach to the design of electrodes for the production of sensors, which show significant changes to the passage of current in response to the concentration of target protein molecules, is presented. Screen-printed platinum electrodes, modified with two separately applied conducting polymer layers, have been developed as a potential route to forming cheap disposable protein sensors. To achieve a heightened response for the target molecules, an initial layer of polypyrrole was formed on the electrode's surface by electro-deposition. This composite was then employed as a substrate for the subsequent electro-deposition of a relatively thin 'sensing layer' of poly-aminophenylboronic acid. Cyclic voltammetry (CV) of the prepared films revealed an excursion in the current versus potential curve in the anodic phase at approximately 0.0 to +0.2V. It was clearly shown that the introduction of proteins into the CV cell resulted in a measurable decrease in the passage of current in buffered aqueous media. Measured current reductions observed on introducing lysozyme (10ppm) into the test solution were 2.3x10(-6)A for an electrode formed with a poly-aminophenylboronic acid layer on platinum, and 1.75x10(-5)A for a composite electrode formed with poly-aminophenylboronic acid on a polypyrrole coated platinum substrate. The introduction of the competing analytes, dl adrenaline or dopamine, at concentrations typically found in human urine, had little effect on the sensor's response. Additionally, the sensing system was able to maintain a response to added target proteins with as much as 2vol.% urine in the test solution. Using the electrodes in high concentrations of competing physiological analytes, they were able to respond to protein concentrations as low as 0.5ppm in buffered solutions containing urea at a concentration representative of human urine (17,000ppm), which additionally contained glucose (1000ppm).     

Recent progress in developing enzyme and tissue-based membrane electrodes

The article summarizes the work of this laboratory on the design of eleven types of amperometricpenzyme electrodes mainly based on the CLARK oxygen sensor in connection with cross-linked oxidases. Special attention is paid to the sensors coupled with plant tissue slice for determining L-ascorbic acid and phenols. A “second generation” bienzyme electrode is reported which enables tow analytes forming a metabolic couple to be determined simultaneously. A principle of the first useful model of conductimetric enzyme electrode for assaying urea and arginase is also presented.     

First results on label-free detection of DNA and protein molecules using a novel integrated sensor technology based on gravimetric detection principles

A novel integrated bio-sensor technology based on thin-film bulk acoustic wave resonators on silicon is presented and the feasibility of detecting DNA and protein molecules proofed. The detection principle of these sensors is label-free and relies on a resonance frequency shift caused by mass loading of an acoustic resonator, a principle very well known from quartz crystal micro balances. Integrated ZnO bulk acoustic wave resonators with resonance frequencies around 2 GHz have been fabricated, employing an acoustic mirror for isolation from the silicon substrate. DNA oligos have been thiol-coupled to the gold electrode by on-wafer dispensing. In a further step, samples have either been hybridised or alternatively a protein has been coupled to the receptor. The measurement results show the new bio-sensor being capable of both, detecting proteins as well as the DNA hybridisation without using a label. Due to the substantially higher oscillation frequency, these sensors already show much higher sensitivity and resolution comparable to quartz crystal micro balances. The potential for these sensors and sensors arrays as well as technological challenges will be discussed in detail.     

Development of a biosensor for urea assay based on amidase inhibition,using an ion-selective electrode

Abstract

A biosensor for urea has been developed based on the observation that urea is a powerful active-site inhibitor of amidase, which catalyzes the hydrolysis of amides such as acetamide to produce ammonia and the corresponding organic acid. Cell-free extract from Pseudomonas aeruginosa was the source of amidase (acylamide hydrolase, EC 3.5.1.4) which was immobilized on a polyethersulfone membrane in the presence of glutaraldehyde; an ion-selective electrode for ammonium ions was used for biosensor development. Analysis of variance was used for optimization of the biosensor response and showed that 30 μL of cell-free extract containing 7.47 mg protein mL^?1, 2 μL of glutaraldehyde (5%, v/v) and 10 μL of gelatin (15%, w/v) exhibited the highest response. Optimization of other parameters showed that pH 7.2 and 30 min incubation time were optimum for incubation of membranes in urea. The biosensor exhibited a linear response in the range of 4.0–10.0 μM urea, a detection limit of 2.0 μM for urea, a response time of 20 s, a sensitivity of 58.245 % per μM urea and a storage stability of over 4 months. It was successfully used for quantification of urea in samples such as wine and milk; recovery experiments were carried out which revealed an average substrate recovery of 94.9%. The urea analogs hydroxyurea, methylurea and thiourea inhibited amidase activity by about 90%, 10% and 0%, respectively, compared with urea inhibition.     

Micro analysis system for pH and protease activities with an integrated sample injection mechanism

A micro analysis system for the electrochemical determination of the activity of protease along with pH sensing was fabricated aiming for its use in telemetric micro analysis systems targeting the testing of the stomach and intestines. The system consisted of a pH-sensing site and two protease assay sites formed in polydimethylsiloxane (PDMS) micro flow channels. To introduce sample solutions, valves were formed with gold electrodes in the inlets, which functioned on the basis of electrowetting. An external sample solution could be introduced into the sensing sites by switching on the valves at appropriate times. In the pH-sensing site, a pH-indicator electrode changed its electrode potential immediately after a sample solution reached an internal liquid-junction reference electrode. The slope of the calibration plot was -74.5 mVpH(-1). Bovine serum albumin (BSA) was used as the substrate for the enzyme and was spotted on the wall of the flow channel that faced the pH-indicator electrode of the protease assay sites. The release of protons accompanying the hydrolysis of BSA by the enzyme was detected using the pH-indicator electrode. When trypsin was contained in the sample solution as a test enzyme, a distinct decrease in pH, which was dependent on the trypsin activity, was observed, indicating that enzymatic hydrolysis was proceeding. The initial rate of potential change varied in proportion to the activity in a range between 1.0 and 51.7 Uml(-1). The integration of the microfluidic and sensing functions provides significant advantages for the use of this system as an isolated telemetric micro system that might operate with small batteries.     

Enantioselectivity of potentiometric sensors with application of different mechanisms of chiral discrimination

In the recent years, numerous successful applications of various chiral selectors in high performance separation methods have generated an increasing interest in the application of some of these compounds as electroactive species in potentiometric sensors. The objective of this work was to examine the enantioselectivy of several different sensors employing substituted cyclodextrins, example antibiotic teicoplanin and electrodeposited conductive polymers for various chiral analytes. Varying degrees of enantioselectivity were found for the ion-selective electrodes examined, depending on the chiral selector used and the target analyte.     

Single-wall carbon nanotube-based voltammetric sensor and biosensor

The pH-sensitive property of the single-wall carbon nanotube modified electrode based on the electroactive group on the single-wall carbon nanotube was explored by differential pulse voltammetry technique. In pH range 1-13 investigated in Britton-Robinson (B-R) buffer, the anodic peak shifted negatively along with the increase of pH exhibiting a reversible Nernstian response. Experiments were carried out to investigate the response of the single-wall carbon nanotube (SWNT) modified electrode to analytes associated with pH change. The response behavior of the modified electrode to ammonia was studied as an example. The potential response could reach equilibrium within 5 min. The modified electrode had good operational stability. Voltammetric urease and acetylcholinesterase biosensors were constructed by immobilizing the enzymes with sol-gel hybrid material. The maximum potential shift could reach 0.130 and 0.220 V for urea and acetylthiocholine, respectively. The methods for preparing sensor and biosensor were simple and reproducible and the range of analytes could be extended to substrates of other hydrolyases and esterases. This broadened the biosensor application of carbon nanotube in electrochemical area.     

Development of potentiometric urea biosensor based on urease immobilized in PVA-PAA composite matrix for estimation of blood urea nitrogen (BUN)

A urea biosensor was developed using the urease entrapped in polyvinyl alcohol (PVA) and polyacrylamide (PAA) composite polymer membrane. The membrane was prepared on the cheesecloth support by gamma-irradiation induced free radical polymerization. The performance of the biosensor was monitored using a flow-through cell, where the membrane was kept in conjugation with the ammonia selective electrode and urea was added as substrate in phosphate buffer medium. The ammonia produced as a result of enzymatic reaction was monitored potentiometrically. The potential of the system was amplified using an electronic circuit incorporating operational amplifiers. Automated data acquisition was carried by connecting the output to a 12-bit analog to digital converter card. The sensor working range was 1–1000 mM urea with a response time of 120 s. The enzyme membranes could be reused 8 times with more than 90% accuracy. The biosensor was tested for blood urea nitrogen (BUN) estimation in clinical serum samples. The biosensor showed good correlation with commercial Infinity™ BUN reagent method using a clinical chemistry autoanalyzer. The membranes could be preserved in phosphate buffer containing dithiothreitol, β-mercaptoethanol and glycerol for a period of two months without significant loss of enzyme activity.     

Insulator semiconductor structures coated with biodegradable latexes as encapsulation matrix for urease

A new urea biosensor for clinical applications was obtained by immobilization of urease within different latex polymers functionalized by hydroxy, acetate and lactobionate groups. Responses of these biosensors based on pH-ion-selective field effect insulator-semiconductor (IS) systems to urea additions were evaluated by capacitance measurements. UV-visible spectroscopy was used to check the urease activity in various matrixes. A good retention of the catalytic urease activity in the case of the cationic polymers was observed. In addition, rotating disk electrode experiments were carried out to determine the matrix permeability characteristics. Under optimal conditions, i.e. buffer capacity corresponding to 5 mM phosphate buffer, the urea enzyme insulator semiconductor (ENIS) sensors showed a linear response for urea concentrations in the range 10(-1.5) to 10(-4)M. Furthermore, kinetic parameters for the immobilized urease were obtained from Lineweaver-Burk plot. Clearly, a fast response and a good adhesion for the urease-acetate polymer composite films, prepared without using glutaraldehyde as cross-linking agent was observed.     

Design and fabrication development of a micro flow heated channel with measurements of the inside micro-scale flow and heat transfer process

The current work provides a design and fabrication technique for a micro channel system that can provide a uniform heat flux boundary condition on the channel wall and a well insulation on the wall to prevent heat loss from the channel to the outside ambient. Therefore, detailed micro-scale flow and heat transfer process and information along the channel can be studied. Semiconductor sensor material was selected to fabricate both the heaters and the arrays of temperature sensors on a silicon substrate. These heaters and sensors were then moved to a low thermal conductivity epoxy-glass substrate for fabrication of the channel. Design consideration and fabrication techniques involved in this processes will be discussed. A final measurement for the validation of the heaters and the sensors fabricated and a study of the flow friction behavior and the heat transfer coefficient distributions inside the micro channel will be presented. The local Nusselt number distrubution inside the micro channel is reported the first time in the open literature.     

Evaluation of the analytical performances of avidin-modified carbon sensors based on a mediated horseradish peroxidase enzyme label and their application to the amperometric detection of nucleic acids

In this study, neutravidin-coated screen-printed carbon sensors were fully characterized and further used for the amperometric detection of specific DNA sequences of human cytomegalovirus (HCMV DNA). For this purpose, we took advantage of an earlier established relationship between the amount of HRP affinity immobilized on the surface of the electrode and the steady-state current recorded in the presence of H₂O₂ as substrate and the single electron donor [Os^III(bpy)₂pyCl]²⁺ as cosubstrate. After incubating a saturating concentration of biotinylated horseradish peroxidase (Bio-HRP) onto the neutravidin-modified sensors, a surface concentration of active HRP of 3.6 pmol cm⁻² was calculated from the measurement of the electrocatalytic plateau current value. This result indicates that monolayers of neutravidin were adsorbed on the screen-printed carbon sensors. These neutravidin-covered platforms were then used to immobilize biotinylated nucleic acid targets. After hybridization with a complementary digoxigenin-labeled detection probe, the extent of hybrids formed was determined with an anti-digoxigenin HRP conjugate. The biosensor assay was applied to the detection of a synthetic oligonucleotide target, and then to the determination of an amplified viral DNA sequence. Monolayers of HRP-labeled oligonucleotide hybrids were immobilized onto the sensing surface whereas one third of the surface was covered with HCMV DNA hybrids. On the other hand, detection limits of 200 pM and 1 nM were obtained for the short oligonucleotide and the longer DNA targets, respectively. Finally, we demonstrated that the sensitivity of the electrochemical assay could be significantly improved by using high concentrations of the reduced form of the mediator [Os^II(bpy)₂pyCl]⁺, thus allowing one to detect as low as 30 pM of amplified HCMV DNA fragment.     

Amperometric enzyme electrodes for aerobic and anaerobic glucose monitoring prepared by glucose oxidase immobilized in mixed ferrocene-cobaltocenium dendrimers

The enzyme glucose oxidase (GOx) has been immobilized electrostatically onto carbon and platinum electrodes modified with mixed ferrocene–cobaltocenium dendrimers. The ferrocene units have been used successfully as mediators between the GOx and the electrode under anaerobic conditions. In experiments carried out in the presence of oxygen, the cobaltocenium moieties act as electrocatalysts in the reduction of the oxygen in the solution, thus making possible the determination of the oxygen variation due to the enzymatic reaction, with high sensitivity. The current response of the electrode was determined by measuring steady-state current values obtained applying a constant potential. The effect of the substrate concentration, the dendrimer generation, the thickness of the dendrimer layer, interferences, and storage on the response of the sensors were investigated.     

Redox labelled avidin for enzyme sensor architectures

Conjugates of avidin with ferrocene and with microperoxidase 8 have been used as electrochemically active molecular building blocks. Assemblies of the conjugates with biotinylated glucose oxidase or lactate oxidase on gold electrodes were tested as enzyme sensors for glucose and lactate. The electrochemical detection is based either on ferrocene-mediated oxidation of the substrate in oxygen-free solution, or on microperoxidase-catalysed reduction of H2O2 which is enzymatically produced from the substrate and molecular oxygen. Glucose and lactate were detectable with both detection principles in concentrations down to 1 or 0.1 mM, respectively. The molecular architecture concept allows quick adaptation of the sensors to other analytes, and it provides a platform for arrays of sensors with different selectivity.     

A disposable biosensor for urea determination in blood based on an ammonium-sensitive transducer

A potentiometric urea-sensitive biosensor using a NH4(+)-sensitive disposable electrode in double matrix membrane (DMM) technology as transducer is described. The ion-sensitive polymer matrix membrane was formed in the presence of an additional electrochemical inert filter paper matrix to improve the reproducibility in sensor production. The electrodes were prepared from one-side silver-coated filter paper, which is encapsulated for insulation by a heat-sealing film. A defined volume of the NH4(+)-sensitive polymer matrix membrane cocktail was deposited on this filter paper. To obtain the urea-biosensor a layer of urease was cast onto the ion-sensitive membrane. Poly (carbamoylsulfonate) hydrogel, produced from a hydrophilic polyurethane prepolymer blocked with bisulfite, served as immobilisation material. The disposable urea sensitive electrode was combined with a disposable Ag/AgCl reference electrode to obtain the disposable urea biosensor. The sensor responded rapidly and in a stable manner to changes in urea concentrations between 7.2 x 10(-5) and 2.1 x 10(-2)mol/l. The detection limit was 2 x 10(-5) mol/l urea and the slope in the linear range 52 mV/decade. By taking into consideration the influence of the interfering K(+)- and Na(+)-ions the sensor can be used for the determination of urea in human blood and serum samples (diluted or undiluted). A good correlation was found with the data obtained by the spectrophotometric routine method.     

Selective glucose detection based on the concept of electrochemical depletion of electroactive species in diffusion layer

A glucose detection approach based on the concept of electrochemical depletion of electroactive species in diffusion layer was established, using scanning electrochemical microscopy (SECM). By controlling the glucose oxidase (GOD) modified electrode (substrate electrode) at a proper potential of electrochemical oxidation of interfering electroactive species, i.e., ascorbic acid (AA), an interference-free microcircumstance was formed in the diffusion layer of the substrate electrode. Consequently, we could successfully sense hydrogen peroxide generated from an enzymatic reaction by locating a Pt ultramicroelectrode (UME) (tip electrode, 5 microm in radius) into the diffusion layer of the substrate electrode. Properties of this interference-removing approach based on electrochemical depletion were systematically investigated. Results showed that the interference-removing efficiency was significantly determined by the tip-substrate distance and substrate potential. When the tip-substrate distance was 11 microm (2.2 times of the tip electrode radius) and the substrate potential was 0.5 V, nearly 90% of AA (0.5 mM) could be depleted within 30s without consumption of H2O2. Under these conditions, 0.1 mM AA showed no influence on the detection of 0.5 mM glucose. The linear range of glucose detection is 0.01-1 mM with a detection limit (DL) of 0.005 mM (correlation coefficient is 0.9948). This research will open a new way for developing selective micro-biosensors.     

The BARC biosensor applied to the detection of biological warfare agents

The Bead ARray Counter (BARC) is a multi-analyte biosensor that uses DNA hybridization, magnetic microbeads, and giant magnetoresistive (GMR) sensors to detect and identify biological warfare agents. The current prototype is a table-top instrument consisting of a microfabricated chip (solid substrate) with an array of GMR sensors, a chip carrier board with electronics for lock-in detection, a fluidics cell and cartridge, and an electromagnet. DNA probes are patterned onto the solid substrate chip directly above the GMR sensors, and sample analyte containing complementary DNA hybridizes with the probes on the surface. Labeled, micron-sized magnetic beads are then injected that specifically bind to the sample DNA. A magnetic field is applied, removing any beads that are not specifically bound to the surface. The beads remaining on the surface are detected by the GMR sensors, and the intensity and location of the signal indicate the concentration and identity of pathogens present in the sample. The current BARC chip contains a 64-element sensor array, however, with recent advances in magnetoresistive technology, chips with millions of these GMR sensors will soon be commercially available, allowing simultaneous detection of thousands of analytes. Because each GMR sensor is capable of detecting a single magnetic bead, in theory, the BARC biosensor should be able to detect the presence of a single analyte molecule.     

Urea sensor with single poly(propylene) membrane

Urease was immobilized on the plasma-aminated surface of a hyfrophobic poly(propylene) (PP) membrane. This membrane, with urease matrix on one side while maintaining its original hydrophobic property on the other, was used to construct the urea sensor. The new urea sensors had response sensitivities ranged from 19 mV/decade to 30 mV/decade depending on the conditions of the plasma reaction. The enzyme electrode using single membrane gave a shorter response time as compared to the corresponding conventional electrode employing two seperate PP membranes. The sensitivity of the enzyme electrode increased with increasing buffer pH and reached a maximal level (40 mV/decade) at pH 7.6. The response sensitivity of the electrode was not affected by the change of buffer strength. Deamination of the plasma-modified hydrophobic PP membrane did not occur in aqueous environment judging from the stability of the urea electrode up to 12 days of operation. (c) 1992 John Wiley & Sons. Inc.     

A thermophilic apoglucose dehydrogenase as nonconsuming glucose sensor

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.     

The development of a 'labeless' immunosensor for the detection of Listeria monocytogenes cell surface protein, Internalin B

Electrochemical impedance spectroscopy (EIS) is a widely used technique for probing bioaffinity interactions at the surfaces of electrically conducting polymers. EIS methods can be employed to investigate 'labeless' detection of analytes via impedimetric transduction. This paper describes the development of a direct immunosensor for the detection of a cell-surface protein on Listeria monocytogenes, an extremely important food-borne pathogen. L. monocytogenes are facultative anaerobic, non-sporing, Gram-positive, motile rods that employ the surface bound protein, Internalin B (InlB), to promote invasion into host cells. A recombinant form of InlB was previously cloned and expressed in Escherichia coli and a panel of antibodies and antibody fragments directed against the protein were also produced. Here, we describe how a portion of the recombinant InlB protein, the F3 fragment, and an anti-InlB polyclonal antibody, were used to develop a platform for the labeless immunosensing of InlB. Sensors were fabricated by electropolymerisation of planar screen-printed carbon electrodes with polyaniline (PANI), to produce a conductive substrate. Polyclonal anti-InlB antibody was subsequently incorporated onto the PANI layer using a biotin-avidin system for site-specific immobilisation. The sensors were then probed with varying concentrations of InlB antigen and the impedimetric response at each concentration was recorded. An anti-IgG antibody was immobilised at the electrode surface, as a control and subsequently exposed to the same concentrations of InlB. Impedimetric data for the control sensors were also recorded. Upon exposure to a range of concentrations of antigen, complex plane impedance analyses were used to relate the differing redox states of the polymer layer, to the possible charge transfer at the surface, with respect to the related mechanisms between the antibody and the polymer. These effects were subsequently monitored to assess the impedance of the polymer thereby determining the amount of bound antigen at the sensor surface. Calibration profiles for both sample (InlB) and control (IgG) sensors were constructed. A limit of detection of 4.1 pg/ml was achieved for Internalin B.     

Biosensor system for continuous flow determination of enzyme activities. I. Determination of glucose oxidase and lactic dehydrogenase activities

A biosensor system for continuous flow determination of enzyme activity was developed and applied to the determination of glucose oxidase and lactic dehydrogenase activities. The glucose oxidase activity sensor was prepared from the combination of an oxygen electrode and a flow cell. Similarly, the lactic dehydrogenase activity sensor was prepared from the combination of a pyruvate oxidase membrane, an oxygen electrode, and a flow cell. Pyruvate oxidase was covalently immobilized on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyloctane, and glutaraldehyde. Glucose oxidase activity was determined from the oxygen consumed upon oxidation of glucose catalyzed by glucose oxidase. Lactic dehydrogenase activity was determined from the pyruvic acid formed upon dehydrogenation of lactic acid catalyzed by lactic dehydrogenase. The amount of pyruvic acid was determined from the oxygen consumed upon oxidation of pyruvic acid by pyruvate oxidase. Calibration curves for activity of glucose oxidase and lactic dehydrogenase were linear up to 81 and 300 units, respectively. One assay could be completed within 15 min for both sensors and these were stable for more than 25 days at 5°C. The relative errors were ±4 and ±6% for glucose oxidase and lactic dehydrogenase sensors, respectively. These results suggest that the sensor system proposed is a simple, rapid, and economical method for the determination of enzyme activities.