Conducting elastomer surface texturing: a path to electrode spotting. Application to the biochip production

A new active support for electro-chemiluminescent biochip preparation has been developed. This material was based on an original material composed of graphite modified polydimethyl siloxane (PDMS). The addressed inclusion of Sepharose beads at the surface of this elastomeric electrode generated interesting local high specific surface. The electrode was characterised by electrochemical (cyclic voltametry, chronoamperomatry) and imaging (scanning electron microscopy (SEM)) methods, and a surface area increase factor of 50 was found, linked to the texturing of the surface generated by the presence of the Sepharose beads. The consequence of this increase was shown to be a jump of the local electrochemical activity which induced a well defined and localised electro-chemiluminescent signal. The new material was used to design biochips based on the electro-chemiluminescent reaction of luminol with enzymatically produced hydrogen peroxide. Thus, when using beads bearing bio-molecules such nucleic acid or human IgG, in conjunction with glucose oxidas-labelled DNA or antibody, sensitive biochips could be obtained with detection limits of 10(11) and 10(10) molecules, respectively. Multi-parameter enzyme-based biochips could also be achieved by locally adsorbing, at the PDMS-graphite surface, either glucose oxidase, lactate oxidase or choline oxidase. Detection limits of 10 microM for lactate and choline and 20 microM for glucose were found, with detection ranging over two decades at least.     

Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh (S/N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.     

Enzyme biosensor for studying therapeutics of Alzheimer's disease.

An electrochemical method for the investigation and comparison of anti-Alzheimer medications that is based on the inhibition of the acetylcholinesterase is presented. The developed amperometric biosensor determines the in-vitro inhibition of the acetylcholinesterase that is co-immobilized with choline oxidase on the working electrode surface of a three-electrode system using gel entrapment. The sensor has been applied to determine the IC50 values of two known and one newly developed Alzheimer remedy. A simultaneous measurement with the photometric standard method shows the applicability of our method for fast drug screening.     

Carbon fibre-based microbiosensors for in vivo measurements of acetylcholine and choline

This report describes technical improvements to the manufacture of a carbon fibre electrode for the stable and sensitive detection of H2O2 (detection limit at 0.5 microM). This electrode was also modified through the co-immobilisation of acetylcholinesterase (AChE) and/or choline oxidase (ChOx) in a bovine serum albumin (BSA) membrane for the development of a sensor for in vivo measurements of acetylcholine and choline. Amperometric measurements were performed using a conventional three-electrode system forming part of a flow-injection set-up at an applied potential of 800-1100 mV relative to an Ag/AgCl reference electrode. The optimised biosensor obtained was reproducible and stable, and exhibited a detection limit of 1 microM for both acetylcholine and choline. However, due to the high operating potential used, the biosensor was prone to substantial interference from other electroactive compounds, such as ascorbic acid. Therefore, in a further step, a mediated electron transfer approach was used that incorporated horseradish peroxidase into an osmium-based redox hydrogel layered onto the active surface of the electrode. Afterwards, a Nafion layer and a coating containing AChE and/or ChOx co-immobilised in a BSA membrane were successively deposited. This procedure further increased the selectivity of the biosensor, when operated in the same flow-injection system but at an applied potential of -50 mV relative to an Ag/AgCl reference electrode. The sensor exhibited good selectivity and a high sensitivity over a concentration range (0.3-100 microM) suitable for the measurement of choline and acetylcholine in vivo.     

Acetylcholine biosensor involving entrapment of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane

A bienzymatic sensor for the determination of acetylcholine was prepared by physical coimmobilization of acetylcholinesterase and poly(ethylene glycol)-modified choline oxidase in a poly(vinyl alcohol) cryogel membrane obtained by a cyclic freezing-thawing process. The enzyme-modified polymer was applied on a platinum electrode to form an amperometric sensor, based on the electrochemical detection of enzymatically developed hydrogen peroxide. The analytical characteristics of this sensor, including calibration curves for choline and acetylcholine, pH, and temperature effects, and stability are described.     

Simple and rapid measurement of acetylcholine and choline by HPLC and enzymatic-electrochemical detection

A simple, rapid and sensitive method for the detection of acetylcholine and choline in tissue extracts is reported. Acetylcholine and choline are first separated by HPLC then react in a mini-column with acetylcholinesterase and choline oxidase immobilized on Sepharose. The resulting H₂O₂ produced by choline oxidase is then detected electrochemically. The assay is more sensitive than existing methods. We believe that the principle involved in this method namely the combination of immobilized enzymes and the high sensitivity of electrochemical detection may be applied to other substances that can be converted by immobilized enzymes into an electrochemically detectable compound.     

Electrochemiluminescent biosensors array for the concomitant detection of choline, glucose, glutamate, lactate, lysine and urate

A multifunctional bio-sensing chip was designed based on the electrochemiluminescent (ECL) detection of enzymatically produced hydrogen peroxide. Six different oxidases specific for choline, glucose, glutamate, lactate, lysine and urate were non-covalently immobilised on imidodiacetic acid chelating beads (glucose oxidase only) or on diethylaminoethyl (DEAE) anion exchanger beads, and spotted on the surface of a glassy carbon foil (25 mm(2) square), entrapped in PVA-SbQ photopolymer. The chip measurement was achieved by applying during 3 min a +850 mV potential between the glassy carbon electrode and a platinum pseudo-reference, while capturing a numeric image of the multifunctional bio-sensing chip with a CCD camera. The use of luminol supporting beads (DEAE-Sepharose) included in the sensing layer was shown to enable the achievement of spatially well defined signals, and to solve the hydrogen peroxide parasite signal which appeared between contiguous spots using luminol free in solution. The detection limits of the different biosensor were found to be 1 microM for glutamate, lysine and uric acid, 20 microM for glucose and 2 microM for choline and lactate. The detection ranges were 1-25 microM (uric acid), 1 microM-0.5 mM (glutamate and lysine), 20 microM-2 mM (glucose) and 2 microM-0.2 mM (choline and lactate). The ECL chip was used for the detection of glucose, lactate and uric acid in human serum matrix. Good correlations between measured and expected values were found without the need of internal calibration of the sample, demonstrating the potentiality of the ECL multifunctional bio-sensing chip.     

Electrochemical biosensors for biocontaminant detection consisting of carbon nanotubes,platinum nanoparticles,dendrimers, and enzymes

The presented approach provides the advanced development of effective, rapid, and versatile electrochemical sensors for a small amount of analytes on potential, cheap, and disposable printed chips. The electrocatalytic activity of this biosensor revealed the feasible detection of hydrogen peroxide at low potential (∼0.09 V) and the detection of a biocontaminant inhibitor (organophosphorus pesticide) in a wide range of concentrations. This efficiency comes from the chemical immobilization of catalysts (Pt nanoparticles) and electron transfer-enlarging materials (carbon nanotubes) on an electrode. Especially, dendrimers raise the stable conjugation of enzymes (acetylcholinesterase/choline oxidase/peroxidase) as well as nanoparticles and carbon nanotubes on an electrode.     

Determination of 3-nitrotyrosine by high-pressure liquid chromatography with a dual-mode electrochemical detector

3-Nitrotyrosine, a product of tyrosine nitration, is useful as a marker for the generation of reactive nitrogen oxide species with short half-lives such as peroxynitrite. A reverse-phase high-pressure liquid chromatographic method using a dual-mode electrochemical detector in series with a photodiode array detector has been developed to determine the levels of 3-nitrotyrosine in biological samples. The principle of this method involves reduction of 3-nitrotyrosine at an upstream gold amalgam electrode and oxidation of the resulting product(s) at a downstream glassy carbon electrode. 3-Nitrotyrosine is quantified by the amount of the current generated at the downstream electrode, and a femtomole detection level can be achieved. The disappearance of the corresponding peak when the electrochemical detector is used only in the single oxidative mode provides additional evidence for the identity of 3-nitrotyrosine in the sample. Tyrosine from the same sample is determined by its UV absorption at 280 nm, thus eliminating the need for an internal standard. With this method a dose-dependent increase of 3- to 10-fold in the levels of protein 3-nitrotyrosine was observed in the blood plasma, and a 2- to 4-fold increase in the lung cytosols, of rats treated with the lung carcinogen and nitrating agent tetranitromethane.     

A Simple, Sensitive, and Economic Assay for Choline and Acetylcholine Using HPLC, an Enzyme Reactor, and an Electrochemical Detector

A simple, efficient, economic, and sensitive method is presented for the detection of choline and acetylcholine in neuronal tissue using HPLC, a postcolumn enzyme reactor with immobilized enzyme, and electrochemical detection. The method is based on a separation of choline and acetylcholine by cation exchange HPLC followed by passage of the effluent through a postcolumn reactor containing a mixture of acetylcholinesterase and choline oxidase; the latter enzyme converts choline to betaine and hydrogen peroxide, the former enzyme hydrolyzes acetylcholine to acetate and choline. The hydrogen peroxide produced is electrochemically detected. A simple and efficient preparation of neuronal tissue is described using an optional prepurification step on Sephadex G-10 columns, offering the possibility to detect choline and acetylcholine as well as catecholamines and their related metabolites in the same tissue sample. The sensitivity of the assay system is 250 fmol for choline and 500 fmol for acetylcholine.     

Development of acetylcholine sensor using carbon fiber (amperometric determination)

An enzyme sensor is developed using carbon fiber to measure acetylcholine concentration. The mechanism is based on the detection of H₂O₂ which is a product of the sequential enzyme reactions of acetylcholinesterase and choline oxidase. The fabrication of the electrode is described. The sensor is polarized at 1.2 V. Enzymes are co-immobilized in polyvinyl alcohol containing styryl pyrydinium (photo-crosslinkable polymer). A fast response time of 0.8 min is obtained. A linear correlation is observed between 0.2 and 1.0 mM. Other optimal operational conditions with respect to pH, temperature and stability are discussed. The use of carbon fiber containing co-immobilized enzymes could offer several model advantages especially in neuroscience research. In conclusion, the aims of the present work are centered on carbon fiber electrode fabrication, immobilization electrochemical measurements.     

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.     

Cobalt(II) salophen-modified carbon-paste electrode for potentiometric and voltammetric determination of cysteine

A chemically modified electrode constructed by incorporating N,N(')-bis(salicylidene)-1,2-phenylenediaminocobalt(II) into carbon-paste matrix was used as a sensitive electrochemical sensor for detection of cysteine. The resulting electrode exhibits catalytic properties for the electrooxidation of cysteine and lowers the overpotential for the oxidation of this compound. The faster rate of electron transfer results in a near-Nernstian behavior of the modified electrode and makes it a suitable potentiometric and voltammetric sensor for the fast and easy determination of cysteine. A linear response in concentration range from approximately 2 microM to 0.01 M was obtained with a detection limit of 1 microM for the potentiometric detection of cysteine. The modified electrode was also used for the amperometric and differential pulse voltammetric determination of cysteine and the results were compared with those of the potentiometric method.     

Comparison of pesticide sensitivity by electrochemical test based on acetylcholinesterase biosensor

Based on the change in electrochemical behavior of enzymatic activity induced by pesticide, a novel electrochemical method has been devised for investigation of pesticide sensitivity using acetylcholinesterase (AChE) biosensor. Because of the excellent biocompatibility and good stability of chitosan matrix, it prevented leakage of the AChE from electrode. Multiwall carbon nanotube (MWNT) promoted electron transfer reaction at a lower potential and catalyzed the electro-oxidation of thiocholine, thus amplifying the sensitivity and amperometric response of the biosensor. Four pesticides of carbaryl, malathion, dimethoate and monocrotophos were selected to discuss their inhibition efficiencies to AChE. The inhibition curves were similar to Michealis-Menten and the Michealis-Menten constants (Km) were calculated to be 0.96 microM, 1.78 microM, 1.97 microM and 4.28 microM, respectively. Ninety-five percent reactivation of the inhibited AChE could be regenerated using pralidoxime iodide within 8 min. The proposed electrochemical pesticide sensitivity test exhibited high sensitivity, low cost and simplified procedures, which is a promising new tool for comparison of pesticide sensitivity and for selection of the most efficient enzyme inhibitors.     

Detection of pesticides using an amperometric biosensor based on ferophthalocyanine chemically modified carbon paste electrode and immobilized bienzymatic system

A new highly sensitive amperometric method for the detection of organophosphorus compounds has been developed. The method is based on a ferophthalocyanine chemically modified carbon paste electrode coupled with acetylcholinesterase and choline oxidase co-immobilized onto the surface of a dialysis membrane. The activity of cholinesterase is non-competitively inhibited in the presence of pesticides. The highest sensitivity to inhibitors was found for a membrane containing low enzyme loading and this was subsequently used for the construction of an amperometric biosensor for pesticides. Analyses were done using acetylcholine as substrate; choline produced by hydrolysis in the enzymatic layer was oxidized by choline-oxidase and subsequently H(2)O(2) produced was electrochemically detected at +0.35 V vs. Ag/AgCl. The decrease of substrate steady-state current caused by the addition of pesticide was used for evaluation. With this approach, up to 10(-10) M of paraoxon and carbofuran can be detected.     

Amperometric and impedimetric characterization of a glutamate biosensor based on Nafion and a methyl viologen modified glassy carbon electrode

An electrochemical biosensor based on a glassy carbon (GC) electrode chemically modified with the perfluorinated cation-exchange polymer Nafion and methyl viologen (MV) is described. The enzyme was immobilized by cross-linking with glutaraldehyde in the presence of bovine serum albumin (BSA), methyl viologen and Nafion. Operating variables such as the enzyme/BSA ratio, cross-linking time in glutaraldehyde vapor, methyl viologen and Nafion percentages were investigated with regard to their influence on the biosensor sensitivity by using glucose oxidase as the enzyme model due to its high stability and low cost. The glutamate biosensor was elaborated by using optimized parameters and its electrochemical properties were investigated by cyclic voltammetry, amperometry and by electrochemical impedance spectroscopy. The glutamate biosensor shows a detection limit of 20 microM and a linear range extended to 0.75 mM. Its selectivity was tested with 15 different amino acids, each with a concentration of 20 microM, 25 microM acetaminophen, 20 microM uric acid and 200 microM ascorbic acid. No amperometric response was observed for the interfering species. This good selectivity allows glutamate detection in biological media without previous separation of the analyte.     

High performance liquid chromatography analysis of a 4-anilinoquinazoline derivative (PD153035), a specific inhibitor of the epidermal growth factor receptor tyrosine kinase, in rat plasma

The quinazoline derivative, 4-N-(3'-bromo-phenyl)amino-6,7-dimethoxyquinazoline (PD153035), has recently been identified as a potential drug for the treatment of proliferative disease. Here, we report a sensitive high performance liquid chromatography (HPLC)-based quantitative detection method for measurement of PD153035 levels in rat plasma. Sample pretreatment involved a two-step extraction with chloroform. The analytes were separated on a column packed with OmniSpher C18 material and eluted with acetonitrile-0.1 M ammonium acetate, pH 7.2 (70:30, v/v). The column effluent was monitored by UV detection at 330 nm. A linear response was achieved over the concentration range 0.50-100.00 microM using multilevel calibration with an internal standard. The analytical method inter- and intra-run accuracy and precision were better than +/-15%. The lower limit of quantification was 0.50 microM. The method has been applied to study the preclinical pharmacokinetics of this compound in rats.     

Assay of acetylcholinesterase activity by potentiometric monitoring of acetylcholine

An acetylcholine-selective electrode based on a plasticized polymeric membrane has been developed. The electrode exhibited good selectivity for acetylcholine (ACh) over choline and some common ions, low drift, and a fast response to ACh. The response was linear over an ACh concentration range of 1×10(-6) to 1×10(-3) M with a slope of 59.1±0.1 and a detection limit of 1.5×10(-7)±1.2×10(-8) M. The electrode was used to monitor enzymatic ACh hydrolysis catalyzed by acetylcholinesterase (AChE) at different substrate and enzyme concentrations. A kinetic data analysis permitted the determination of the Michaelis-Menten constant of the enzymatic hydrolysis and AChE activity in the range of 2×10(-5) to 3.8×10(-1)U ml(-1).     

Measurement of acetylcholine and choline in brain by HPLC with electrochemical detection

A simple and rapid method for measuring acetylcholine and choline using high performance liquid chromatography (HPLC) with electrochemical detection is presented. Acetylcholine and choline were first separated using reverse-phase chromatography; acetylcholine was then hydrolyzed post-column to choline by acetylcholinesterase. Choline was oxidized enzymatically by choline oxidase to betaine and hydrogen peroxide, and the peroxide was detected electrochemically. Changes in methodology from previous procedures include a different mobile phase, controlled heating of chromatography column and post-column reaction coil, and a different extraction method for quaternary amines. The changes resulted in less inhibition of derivatizing enzymes by mobile phase, narrow and consistent elution of peaks, and a rapid and efficient extraction of quaternary amines. Measurement of acetylcholine and choline in brain tissue was found to be replicable, and the levels agreed with literature values.     

Determination of serum cholinesterase activity by liquid chromatography with electrochemical detection.

A sensitive enzymatic assay to measure cholinesterase activity in serum using liquid chromatography with electrochemical detection has been devised and used to examine cholinesterase inhibition in mice treated with diisopropyl phosphorofluoridate. Acetylcholine was used as substrate, and a postcolumn reactor containing immobilized choline oxidase converted the enzymatic product, choline, and the internal standard, ethylhomocholine, into the electrochemically active H2O2. The postcolumn reactor also contained acetylcholinesterase to allow the indirect detection of the substrate. Assay optimization included investigations of substrate concentration, buffer pH and ionic strength, enzyme concentration, incubation time, and reaction termination method. The optimized procedure is applicable to samples with activities of 0.11 to 269 mmol/ml/h. Intrasample coefficient of variation for mouse serum samples was 1.7% (n = 12), while intersample coefficient of variation was 8.0% (n = 5). The mean +/- SE serum cholinesterase activity found for controls and mice treated with diisopropyl phosphofluoridate (6.3 mg/kg, ip, 24 h prior) was 158.7 +/- 5.7 mumol/ml/h and 36.6 +/- 3.1 mumol/ml/h, respectively (P less than 0.001).