Biomagnetic neurosensors.

Non-invasive measurement of the neuromodulatory activity of certain analytes is now possible through the use of biomagnetic stimulation and detection techniques. The timely development of room-temperature instrumentation and of more effective techniques for coupling neurons to transducers are the critical elements for rapid progress in this field.

Biomagnetic neurosensors. 4. Design and optimization for analytical use.

Biomagnetic neurosensors based on magnetic stimulation and magnetic detection of neural events depend critically upon the effective matching of the magnetic transducers and the neural tissue employed. Although the properties of wire-wrapped ferrite core transducers can be predicted from electromagnetic fundamentals, meaningful analytical measurements using real nerves as molecular recognition elements require additional calibration and optimization steps in order to achieve good system response and lifetimes. This note provides some design guidelines and experimental test procedures to enable potential users to employ biomagnetic neurosensors in other laboratories.

Biomagnetic neurosensors. 3. Noninvasive sensors using magnetic stimulation and biomagnetic detection.

A noninvasive biomagnetic sensor system that uses magnetic toroids for both neural stimulation and detection is described. It is shown that analytical signals obtained by direct magnetic detection (no signal averaging) compare favorably with electrical monitoring and that dose-response curves for local anesthetics correlate well between the two methods. Neural lifetimes are significantly extended when the noninvasive biomagnetic sensing system is used.

Biomagnetic neurosensors. 2. Magnetically stimulated sensors.

We report the generation of action potentials in crayfish neurons by magnetic pulses for analytical purposes. A copper wire toroid, containing a ferrite core, was placed around the nerve bundle, and square wave current pulses were sent through the wire to generate a magnetic field. The magnetic field generated an action potential in the neuron that was detected downfield by a pickup microelectrode. The biosensor was used to detect local anesthetics by monitoring the time necessary for complete blockage of the action potential. Techniques for improving the efficiency and lifetimes of neural biosensors are discussed.

Simultaneous determination of lactose and glucose in milk using two working enzyme electrodes.

Lactose and glucose concentrations were determined simultaneously by using a measuring-cell containing lactose and glucose electrodes made by mixing beta-galactosidase/glucose oxidase and glucose oxidase, respectively, with carbon paste. The glucose electrode responds to glucose alone, while the lactose electrode measures the sum of glucose and lactose. Lactose concentration was calculated by subtracting the glucose concentration from the reading of the lactose electrode. The present dual-working electrode system permitted the determination of lactose and glucose concentrations simultaneously from a single measurement over a linear range of 0.1-2.5 mM. Furthermore, it enabled the determination of lactose concentration in milk in the presence of glucose to be carried out more precisely and with a higher degree of sensitivity than the conventional calorimetric method.

Biomagnetic neurosensors.

In this report we demonstrate the first analytical application of biomagnetic field detection at nerve fibers for biosensing purposes. A ferrite core toroid surrounding the nerve, coupled to a low-noise, low-input-impedance amplifier, is used to inductively detect the compound action current (CAC) in crayfish giant axons upon stimulation of nerve firing. Detection of the local anesthetic lidocaine, which blocks neuronal conduction by binding in the ion channel of the voltage-gated sodium channel receptor, is achieved by monitoring the disappearance of the CAC. The application of this novel detection principle to the screening of neurotoxic and neuromodulatory drugs and natural product extracts is proposed.

Toxin detection using a tyrosinase-coupled oxygen electrode.

An enzyme-based "electrochemical canary" is described for the detection of cyanide. The sensing system imitates cyanide's site of toxicity in the mitochondria. The terminal sequence of electron transfer in aerobic respiration is mimicked by mediator coupling of tyrosinase catalysis to an electro-chemical system. An enzyme-coupled oxygen electrode is created which is sensitive to selective poisoning. Biocatalytic reduction of oxygen is promoted by electrochemically supplying tyrosinase with electrons. Thus, ferrocyanide is generated at a cathode and mediates the enzymatic reduction of oxygen to water. An enzyme-dependent reductive current can be monitored which is inhibited by cyanide in a concentration-dependent manner. Oxygen depletion in the reaction layer can be minimized by addressing enzyme activity using a potential pulsing routine. Enzyme activity is electrochemically initiated and terminated and the sensor becomes capable of continuous monitoring. Cyanide poisoning of the biological component is reversible, and it can be reused after rinsing. The resulting sensor detects cyanide based on its biological activity rather than its physical or chemical properties.

Biosensors based on plant and animal tissues.

A biosensor is defined as a device that incorporates a biological component which is either intimately connected to or integrated within a transducer. Biocatalysts, such as specialized tissues from higher animals and plants, have been incorporated into various electrochemical transducers to construct biosensors for the detection of important analytes. The receptor-based biosensors utilize isolated receptors or whole intact receptor organisms as molecular recognition elements for the detection of various important analytes including drugs, hormones, toxicants, neurotransmitters and amino acids. The immobilization of isolated receptors on transducers such as ISFETs, electric capacitors and optical fibers for biosensing has been given significant consideration recently. Intact chemoreceptor-based biosensors, in particular, offer several advantages including extremely short response time, a high degree of sensitivity, a wide range of linear response and inherent selectivity. This review highlights some of the recent advances in plant and animal tissue-based biosensors, with emphasis on historical developments.

Microtiter plate binding assay for cholinergic compounds utilizing the nicotinic acetylcholine receptor.

A receptor-based binding assay for the determination of cholinergic compounds of the nicotinic acetylcholine receptor has been developed. By conducting the assay in a 96-well microtiter plate, the method is suitable for large-scale screening in drug development. Solid-phase extraction of the enzyme label significantly simplifies the assay protocol compared to earlier methods. The assay is based on immobilization of biotin-BSA on the microtiter which takes up avidin-labeled peroxidase due to avidin-biotin interaction. To perform the assay, a ligand (the analyte) and a biotin alpha-bungarotoxin conjugate (alpha Bgt-biotin) sequentially bind to a vesicle bound nicotinic acetylcholine receptor. This is done either in a test tube, assay I, or in a biotinylated microtiter well, assay II. Avidin-HRP is then added to this mixture; free alpha Bgt-biotin conjugate and immobilized biotin-BSA compete for the avidin sites. After the assay solution has been aspirated off, bound enzyme activity is determined which is directly related to the amount of alpha Bgt-biotin added. Dose-response curves of cholinergic compounds and Scatchard plots were generated to evaluate the apparent binding constants. Kinetic studies were conducted for the purpose of optimization. The final assay can be performed in under 4 h with a minimum of sample handling.

Flavonoid-specific staining of Arabidopsis thaliana.

Crop yields may be threatened by increases in UV-B radiation resulting from depletion of the ozone layer. In higher plants, the presence of flavonols provides a protective mechanism, and we report a novel staining procedure for the visualization of such protectants in plant tissue. It is shown that the proposed technique provides sensitive and specific fluorescence of flavonoids in chlorophyll-bleached tissue of Arabidopsis thaliana.

Biotinylated 1012-S conjugate as a probe ligand for benzodiazepine receptors: characterization of receptor binding sites and receptor assay for benzodiazepine drugs.

A nonisotopic receptor assay using the biotin-1012-S conjugate was developed and the usefulness of this conjugate as a probe ligand for the benzodiazepine receptor was evaluated. The conjugate was incubated in a receptor suspension, and then the concentration of free conjugate in the supernatant was determined nonisotopically with a solid-phase avidin-biotin binding assay. Studies on the ligand saturation with the conjugate demonstrated that the conjugate has very high affinity and specificity for the receptors and the biotin labeling does not decrease the affinity of 1012-S. This assay method was applied to the characterization of binding sites of benzodiazepine receptors in cow brain. Competition interactions between the conjugate and benzodiazepine drugs gave well-defined dose-response curves. These results confirm the possibility that this conjugate could serve as a probe for the study of receptor-ligand interactions and provide the basis of a new nonisotopic receptor assay for benzodiazepine drugs.

Non-isotopic receptor assay for benzodiazepines using a biotin-labeled ligand and biotin-immobilized microtiter plate.

A non-isotopic receptor assay for benzodiazepine drugs was developed using a biotin-labeled ligand, biotin-1012S. Biotinylated bovine serum albumin (biotin-BSA) was immobilized onto the wall of microtiter plate wells by simple adsorption. Avidin peroxidase conjugate could be extracted from solution owing to its strong interaction with biotin. The amount of avidin peroxidase taken up on the wall was then determined by measuring the enzyme activity. The competition between immobilized biotin on the wall and free biotin for avidin provided the basis for a solid-phase avidin-biotin binding assay. By this binding assay, not only biotin but also biotin-1012S could be measured sensitively. Because 1012S is a ligand with high affinity to benzodiazepine receptors, biotin-1012S could be utilized as a probe ligand for a non-isotopic receptor assay. Based upon the competition between biotin-1012S and various benzodiazepine drugs for the receptor binding sites, a non-isotopic receptor assay was demonstrated.

Reagentless enzyme electrode for the determination of manganese through biocatalytic enhancement.

An amperometric enzyme electrode is described for the detection and determination of manganese(II). The biosensor is based on the stimulation by manganese of the aerobic oxidation of substrates by horseradish peroxidase. A mediator, 1,2-naphthoquinone, is used as the substrate and is incorporated with the enzyme into a carbon-paste electrode. The resulting electrode acts as an enzyme-based oxygen sensor, which is sensitive to manganese. Electrochemical control of enzyme activity is achieved through substrate promotion of catalysis. Enzyme modulation by manganese can be switched on and off or adjusted through the appropriate selection of the applied potential. Currents are generated due to the bioelectrocatalytic reduction of oxygen in response to the introduction of manganese sulfate. A sustained current is achieved which is dependent on manganese concentration. Concentrations of 0.5 microM manganese or greater can be measured, and the sensor is reversible, as demonstrated by manganese removal. Biological selectivity for manganese provides a sensor which does not respond to other divalent cations tested, with the possible exception of cobalt. Reagentless, continuous sensing is achieved through substrate cycling.

Nonisotopic receptor-binding assay for benzodiazepine receptors utilizing a fluorophore labeled ligand.

A nonisotopic receptor-binding assay method provides a new approach for the study of receptor-ligand interactions and a possible receptor assay for benzodiazepine drugs. The proposed method is based upon the use of fluorescence-labeled drugs and a chromatographic system which accepts samples without deproteinization. The effectiveness of the technique is illustrated in a study of benzodiazepine receptor-drug-binding interactions.

Intact chemoreceptor-based biosensors.

A receptrode biosensor is presented that uses intact chemoreceptor-based molecular recognition from antennular structures of the Hawaiian swimming crab species Portunis sanguinolentus. The sensor is coupled to a learning, pattern recognition calculation for performing analytical chemistry. Action potential waveforms are used to establish the identity of individual action potential types that can be associated to particular analytes. The pattern recognition calculations used are referred to as cluster analysis (CA) and principal component analysis (PCA). Action potential similarities are determined by using a dendrogram plot of the cluster analysis results and further elucidated by using principal component scores plots. Quantitative analysis was performed after classification of analyte and background responses. Chemoresponses to salinity and trimethylamine N-oxide, two chemical constituents that are found in the crustacean living environment, were investigated and gave analytic responses over several orders of magnitude.

Enzymatic solid-phase assay for biotin and a biotin-benzodiazepine conjugate.

A novel enzymatic ligand binding assay for biotin and its benzodiazepine conjugate is based on their binding to horseradish peroxidase-avidin conjugate (A-P) followed by the uptake of biotin-unsaturated A-P onto polystyrene beads coated with biotin-BSA. The detection limit is 1.3 x 10(-16) mol per tube (300 microL) with a 3.3 x 10(-12) M A-P solution and varies with the conjugate concentration employed. The coefficient of variation for 10 repetitive assays of 10(-15) mol of biotin is 6.22%.

Enzyme-amplified receptor assay screening test for chlorpromazine, trifluoperazine, and phencyclidine.

A new receptor based assay is described for the determination of classes of drugs which have high affinities for the acetylcholine receptor. The method is based upon the inhibition of the enzyme activity of an enzyme-drug conjugate by the binding to the receptor protein, and competition between free drugs and the enzyme-drug conjugate for a limited number of receptor sites. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate of NADH formation at 340 nM. The procedure proposed is designed to provide a simple drug screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for three acetylcholine channel binding compounds: the hallucinogen phencyclidine (PCP) and the antipsychotic agents chlorpromazine and trifluoperazine. This procedure yields calibration curves with detection limits at nanomolar levels of drug, with binding responses dependent on the amounts of receptor and enzyme-labeled drug used. Aspecific binding responses of unlabeled enzyme to drug or receptor to compounds with low affinity for the receptor are shown to have minimal effect on the assay.

Conductometric transducers for enzyme-based biosensors.

The use of alternating current conductometric transducers in biosensing devices has been investigated for urea and D-amino acid sensors using the enzyme systems urease and D-amino acid oxidase/catalase. Transducers with copper and platinum electrodes were constructed and characterized, and two enzyme immobilization methods were tested. Detection limits of 1 x 10(-6)M and linear ranges of 2 orders of magnitude were routinely achieved for these model sensors with enzymes covalently immobilized on collagen films.