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An ion pair mechanism is used to characterize the inhibition by DNA, and sensitization by acridine orange (AO) of the peak currents in the cyclic voltammetry (CV) of naphthoquinones with differing ionic substituents. Gold electrodes modified with adsorbed synthetic ′5-thiol-(GC)10 double strand DNA oligomers are used to study the redox activity of ferricyanide, 1,2-naphthoquinone-4-sulfonic acid sodium salt (NQS), 4-amino-1,2-naphthoquinone (ANQ), or 1,2-naphthoquinone (NQ) in the presence of AO. Parallel studies are performed using bare gold electrodes with double strand calf thymus DNA (CT-DNA) in the bulk solution. AO is known to bind intercalatively to DNA and is seen to sensitize the redox activity of ferricyanide or NQS with DNA either adsorbed on the electrode or present in the electrolyte. AO otherwise does not sensitize the peak currents in the CV of either ANQ or NQ regardless of the presence of DNA, nor does the DNA inhibit their CV, as it does the AO sensitized CV of the anions in the presence of DNA. The electroanalysis is done in pH 7 phosphate buffer with the solute and calf thymus double strand DNA base pair concentrations in the 10–3 molar range, and AO concentrations in the 10–4 molar range. The anion selective electrolytic sensitization combined with the intercalative property of AO may be applied to either the design of DNA based biosensors for the detection of anions, or DNA sensitive biosensors using AO as a marker.

Self-assembly of a hapten monolayer is used for the development of amperometric and piezoelectric immunosensors. A disulfide compound carrying a terminal pyrene group was newly synthesized for the modification of a gold surface. The self-assembled organic monolayer was formed spontaneously through the use of a disulfide head group. The resulting monolayer and its long-term stability on a gold surface were measured by cyclic voltammetry. The application of the hapten monolayer to amperometric and piezoelectric immunosensing of monoclonal antibodies and benazo[a]pyrene (BaP) was also investigated.

Neuronal biosensors are employed to assess the effect of selected anti-HIV drugs on nerve tissue. Preliminary experimental results show that Zalcitabine and Didanosine produce interference in neural function and suggest a correlation with clinically observed neuropathy. Zidovudine (AZT) produced no observable effect.

A piezoelectric immunosensor has been developed for the detection of various polycyclic aromatic hydrocarbons (PAHs). The antigen (benzo[a]pyrene–BSA conjugate, BaP–BSA) was immobilized through thioctic acid on gold coated quartz crystals, with a basic resonant frequency of 10 MHz. The monoclonal antibody 10c10 (mAb10c10), which specifically recognizes benzo[a]pyrene (BaP), was detected through frequency decreases (mass increase due to the immunoreaction). Determination of BaP was performed in the flow injection system by using a competitive pattern, in which BaP reacted with the bound mAb10c10 causing frequency increases. A nanomolar level of BaP can be detected with this immunosensor. The mAb10c10 modified quartz crystal cross-reacts with pyrene and naphthalene.

A capacitive immunosensor for the detection of the interaction between benzo[a]pyrene-bovine serum albumin (BaP-BSA) and a monoclonal antibody (designated as MAb10c10) specific to BaP has been developed. MAb10c10 was immobilized on a gold electrode through a self-assembled monolayer of cystamine. Linear sweep voltammetry showed that the charging current decreased with BaP-BSA binding to the antibody on the electrode, and calibration curves were constructed for BaP-BSA and Pyrene-BSA in the range of 0.01 μM-6.00 μM. Selectivity over structurally related analytes is observed.

Information about the kinetics of reactions can readily be extracted from linear sweep voltammograms of adsorbates which show irreversible behavior if the adsorbates behave ideally. Factors which cause nonideal behavior include lateral interactions between adsorbates, influence of solvent reorganization energies, and kinetic dispersion. The difficulties in extracting information on the kinetics of adsorbed redox systems which behave nonideally are discussed. Diagnostic criteria are provided from which it may be determined which of the above phenomena, if any, are effecting the voltammetric response.

An electrochemical biosensor for the detection of organophosphorus pesticides is described. A tyrosinase-based oxygen sensor is constructed where enzymatic oxygen consumption is monitored electrochemically with the mediator 1,2-naphthoquinone-4-sulfonate. This bioelectrocatalytic system allows electrochemical initiation and termination of the enzymatic reaction. Amperometric and coulometric techniques are used to study inhibitory effects for pesticide determinations. This inhibition appears to be fully reversible, with full catalytic activity returning after removal of the pesticides. Detection limits are 5 μM for diazinon and 75 nM for dichlorvos. Enzymatic inhibition of 50% occurs at ∼1000 and ∼50 μM, respectively.

The use of liposomal drug incorporation significantly improves the sensitivity of neuronal sensors to poorly soluble drugs and extends the applicability of such sensors to previously undetectable compounds. Data presented support physiological models for liposome−neuron interactions and are consistent with the channel-blocking actions of local anesthetics. We have incorporated the anesthetics benzocaine, bupivacaine, and tetracaine into liposomes, generating dose−response curves. Liposomes are shown to extend the range of the biosensor and improve its sensitivity.

In this study, the response characteristics for a dual-enzyme fiber-optic biosensor for pyruvate are presented. An enzyme layer composed of lactate oxidase and lactate dehydrogenase (LDH) was fabricated at the tip of a sensing optrode. The biosensor is based on the luminescence detection of consumed reduced nicotinamide adenine dinucleotide (NADH) and the measured fluorescence intensity is related to the bulk concentration of pyruvate. Such a dual-enzyme optrode can not only retain the merits of fiber-optic biosensors, but can also take advantage of two enzyme-catalyzed reactions. The response sensitivity and detection limit of the proposed dual-enzyme sensors are improved by at least 8 and 5 times, respectively, compared to those of single-enzyme systems.

A theory is presented to account for the characteristics of cyclic voltammograms (CVs) at monolayer covered electrodes. It is shown that CVs of reversible systems can exhibit peak separations of gr...

There is a world-wide interest in screening terrestrial and marine plants for components having medicinal properties or prototypes for rational drug design. Hawaiian plants represent a special rich source because many species that grow in Hawaii grow nowhere else. Conventional analytical techniques are of limited utility in this connection because the demonstration of the desired medicinal activity must precede the identification and isolation of the responsible agent. This situation is particularly apparent in the case of neuromodulatory agents whose effect may manifest itself through very diverse modes of action. In this paper, we describe a relatively uncomplicated screening system, derived from biosensor technology, which combines neural tissue as the target material with the electrochemical detection to monitor and quantify neuromodulatory activity. It will be seen that the effects of the neuroactive agents in some Hawaiian plant materials can be demonstrated and evaluated for relative potency.

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.

We describe a novel pyruvate selective sensor in which corn tissue, as the biocatalytic layer, is coupled to a fluorescent indicator-based fiber-optic CO2 optrode. This sensor showed shorter response times and a lower detection limit without loss of enzymatic stability as compared to a corn tissue-based pyruvate selective membrane electrode previously studied.

The theoretical basis and experimental evidence for a Stern-Volmer type relationship using the fluorescence ratio (I bl /I) in the absence (I bl ) and presence (I) of CO 2 as response function for fluorescence-based fiber-optic CO 2 sensors is explored. By choosing the right indicator, appropriate bicarbonate concentration, and a thin enough sensing layer, a fiber-optic CO 2 sensor with a usable linear range can be easily achieved using I bl /I as the response function. This allows the possibility of one-point calibration, which cannot be accomplished if the measured fluorescence intensity (I) is used directly as the response function.

An amperometric biosensor for the determination of pectin was constructed by combining orange peel tissue as a pectinesterase source with a carbon paste alcohol electrode using alcohol oxidase. A slice of orange peel attached to the surface of the alcohol electrode deesterified pectin, the methanol generated by the reaction was monitored by the electrode. A linear response to pectin was observed in the concentration range of 0.1 to 0.9 g l −1 . No interference from other carbohydrates was observed.

A putrescine biosensor has been developed. Putrescine oxidase, peroxidase, and bovine serum albumin were cross-linked directly on the surface of a graphite electrode by glutaraldehyde. The sensor was used to determine putrescine at 0 mV (vs. Ag/AgCl reference electrode) in a potassium phosphate buffer (pH 7.4) solution containing 4 mM dopamine (3-hydroxytyramine). The response of putrescine was linear up to 600 μM with a standard deviation of 2.8%. The detection limit was 5 μM. The response time was around 30 s. The lifetime was over 30 days when the electrode was used every day. The interferences of cadaverine and spermidine could be eliminated by added dopamine which, apparently, inhibits the enzymatic conversion of these substrates more strongly than that of putrescine.

γ-Aminobutyric acid (GABA) was determined by using flow injection analysis with a column of GABAse immobilized on glass beads. The NADPH produced was monitored fluorometrically. At a flow rate of 0.8 ml/min, peak heights were linear in the range 1-500 μM, and samples could be injected every minute. Nearly 100 % recovery of GABA was observed when GABA was added to infusions of green tea. ∗Permanent address: National Research Institute of Vegetables, Ornamental Plants and Tea, 2769 Kanaya, Shizuoka 428 JAPAN

A dual-working electrode (K1 and K2) multienzyme sensor, made of carbon paste, glucose oxidase (GOD) membrane (on K1) and invertase (INV)/mutarotase (MUT)/GOD membrane (on K2), is proposed for simultaneous determination of glucose and sucrose. Interference to the K2 caused by H2O2 produced in the enzyme reaction on K1 can be eliminated by depositing a catalase (CAT) layer between K1 and K2. Acceleration of glucose mutarotation by MUT was found to be three times faster than by phosphate buffer. Glucose was determined directly from the response of K1, while sucrose was computed based on a formula derived from a two-point calibration method. The maximum concentration of sucrose that could be determined depended strongly on the concentration of coexisting glucose. The activities of immobilized GOD, INV, MUT, and CAT were examined individually and found stable at room temperature for at least 1 month. The final loss of sucrose response activity was found to be due to denaturation of INV.

A new amperometric biosensor for adenosine-5′-triphosphate (ATP) was designed using a platinum-dispersed carbon paste into which glycerol kinase and glycerol-3-phosphate oxidase were incorporated. The biosensor is based on the detection of hydrogen peroxide produced by the enzymatic reaction of ATP with glycerol and the subsequent oxidation of glycerol-3-phosphate. The use of the platinum-dispersed carbon paste electrode lowered the oxidation potential for hydrogen peroxide, permitting the sensitive detection of ATP at 0.4 V vs. Ag/AgCl. A linear response to ATP was observed in the concentration range of 1 x 10−5 to 2.5 x 10−3 M. Permanent address: Faculty of Pharmaceutical Sciences, Okayama University, Tsushima, Okayama 700, Japan.

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.

Genetically modified plant tissue materials offer possible advantages as molecular recognition elements in biosensor design. A prototype transgenic biosensor, using potato tissue transformed with a gene confering β-glucuronidase (GUS) biocatalytic activity coupled with fluorescence detection, is described and evaluated. Under optimal operating conditions, the transgenic sensor system gives good response to glucuronide substrate in the micromolar range and has an operating lifetime of at least 2 months at room temperature. Parallel experiments with nontransformed potato plant tisue show no glucuronidase activity. The prototype system described here illustrates that desired pathways can be created in plant tissue through genetic manipulation of higher plants normally lacking such pathways

A bioelectrochemical system for the sensing of benzoic acid is described. An enzyme-based oxygen electrode is constructed where the enzymatic consumption of oxygen by tyrosinase is monitored electrochemically through the mediator 1,2 naphthoquinone, 4-sulfonate (NQS). The bioelectrocatalysis of oxygen can be electrochemically initiated and terminated and is sensitive to inhibition by benzoic acid in a concentration-dependent manner. Inhibition of catalytic current results from a competitive interaction of benzoic acid at the enzymatic docking site of NQS. This inhibition of catalytic activity is reversible, and full catalytic activity can be reinstated following the removal of benzoic acid from solution. The sensing of benzoic acid is dependent on the nature of the mediator used, occurring with NQS but not with ferricyanide. These studies provide support for the possibility that analytes can be targeted for detection in such inhibition-based sensing through the proper selection of mediator.

An image analyzer system was used to develop a multicomponent analysis method for enzyme substrates. Image analysis provides a technique which can spatially resolve closely spaced responses, thus allowing for the detection of multiple substrates with a single reactor/detector. For successful implementation of this method a procedure for immobilizing large amounts of enzyme activity onto a highly crosslinked polydextran support (⪢300 U/ml gel) was devised. A microsized integrated reactor/detector (

This report demonstrates the use of the nerve bundle from the crayfish walking leg for the detection and assay of local anesthetics (LAs). Detection is achieved by monitoring the disappearance of the compound action potential (CAP) upon application of the local anesthetics to the nerves, pinned in a specially designed flow cell. CAP firing is induced with an electrical current pulse applied to the nerve bundle. Dose-response curves for the LAs tetracaine, lidocaine and procaine are constructed and factors which affect the detection are discussed. The application of this procedure to the screening for neurotoxicity and neuromodulatory activity of new drugs and natural product extracts is proposed.

Neuronal biosensors based on the use of crayfish Procambarus clarkii walking legs are reported for the first time and possible analytical applications are explored. The neuronal biosensor is shown to respond selectively to the antitubercular drug and potent food marker, pyrazinamide. The sensor is characterized with respect to selectivity, dose-response relationship, reproducibility and operating lifetime. The detection of local anesthetics is accomplished by monitoring the decrease in pyrazinamide-induced nerve firing in the crayfish walking leg. This decrease is a result of the local anesthetic block of the axonal sodium channels in the nerves. Applicability of this novel procedure to analytical measurements is demonstrated by the construction of dose-response curves for several local anesthetics. Problems associated with the current sensor configuration are presented and future research directions that may improve the neuronal-based biosensor are discussed.

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.

A review of the progress of research in the development of intact chemoreceptor-based biosensors is presented. These sensors utilize the chemosensing ability of the antennules of various decapod crustacea to sensitively and selectively detect chemical stimulants. Suction microelectrodes attached to the antennular nerve fibers are used to detect action potential firing upon binding of the stimulant to the receptor, allowing quantitation of the analyte. Recent developments in the use of different species and in improvements in experimental setup, data handling procedures, and sensor lifetimes are reported.

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.

A nonisotopic receptor based assay is developed which is centered upon the α-toxin-binding nicotinic acetyl-choline receptor and fluorescein labeled α-bungarotoxin. The assay method is based on sequential saturation of the receptor by unlabeled and fluorophore labeled ligands, followed by separation of the free from the bound label by centrifugation. Subsequently, the free concentration is determined by use of a flow injection system. Unlabeled ligands investigated include both the agonist and antagonist types that bind to the receptor α-subunits, i.e., α-bungarotoxin, carbamylcholine, decamethonium, gallamine, d-tubocurarine, pancuronium, and hexamethonium. The effectiveness of the proposed method is illustrated by Scatchard analysis, Hill plot of the labeled ligand, and displacement curves for unlabeled ligands.

Development of a reusable neuronal sensor selective to the neurotoxin 3-acetyl pyridine is reported. The antennular structure from fresh water crayfish (Procamburus clarkii) was used as a sensing element to measure the frequency of action potential responses evoked by chemical stimulation of nerve cells. Reusability of the sensor was studied with emphasis on the reproducibility of action potential responses. Characterization of the novel sensor in terms of selectivity, response time and dose/response relationship is reported. Current status of lifetime extension and future directions of neuronal biosensor research are discussed.

Oat seedling tissue and ferrocene/ferricinium were coimmobilized in a carbon paste matrix to construct a biosensor for polyamines. The oat tissue is rich in the enzymes polyamine oxidase and peroxidase while the mediator facilitates electron transfer between the electrode and hydrogen peroxide. The determination of the polyamines spermine and spermidine, in both batch and flow injection modes, was evaluated. The concentration of polyamine was monitored through hydrogen peroxide in the presence of oxygen as cosubstrate of polyamine oxidase. A reductive potential of 0.0 V was chosen for monitoring ferricinium which is generated by peroxidase. The detection limit (S/N = 3) for spermidine and spermine in the batch system was 0.19 and 1.15 μM, respectively. The relative standard deviation for twelve replicate analyses of 10 μM spermidine was 4.5%. The activity of the tissue modified carbon paste electrode decreased to 45% of the original value after a storage for 8 days in buffer solution at 4°C.

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.

The use of a novel thin layer electrode design, with a plant tissue reactor and a sensing tin oxide collector electrode, is proposed for amperometric detection in flow injection analysis and compared with previous carbon paste electrode designs. The new system, evaluated for the case of catechol measurements, is shown to offer superior sensitivity, dynamic range and detection limits.

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.

In vitro cultured tobacco callus tissue, which contains a high activity of peroxidase, was used to construct an amperometric biosensor for the detection of hydrogen peroxide. The tissue was incorporated into a carbon paste matrix along with ferrocene, an electron mediator. The detection of hydrogen peroxide was accomplished through its enzymatic reduction mediated by ferrocene. Comparison studies conducted with normally grown types of tobacco tissues indicate that the callus tissue has highest biocatalytic activity and the least variance between tissue preparations with respect to sensor response toward hydrogen peroxide. Additionally, the sensor exhibits a remarkably along lifetime of more than 5 months. Other important characteristics of the sensor include fast response times (less than 2 s), wide dynamic range (5 × 10 −6 -1.10 × 10 −4 M) and a detection limit of 7.5 × 10 −7 M hydrogen peroxide.

Receptor-based biosensors using structures from fresh-water species (crayfish) are shown to give selective analytical responses to the antitubercular drug pyrazinamide. The construction of such biosensors, in general, is greatly facilitated through the use of a video imaging inverted biological microscope during the manipulation and assembly of the delicate structures involved and points the way toward microscale receptors with enhanced analytical properties in the future. Characterization of the sensor in terms of selectivity, sensitivity, response time, dose—response relationship and lifetime is reported. Advantages of using fresh-water species in constructing neuronal biosensors, e.g., extended lifetime and fewer interferences, are also discussed.

A new receptor assay method for benzodiazepine was developed using biotin-1012S conjugate as a non-isotopic ligand. The concentration of free ligand in the benzodiazepine receptor suspension was determined by an enzymatic solid-phase avidin-biotin binding assay. The competition reaction between the conjugate and diazepam gave a well-defined dose/response curve for diazepam. The receptor assay could also be applied to the determination of an inverse agonist, n-butyl-P-carboline-3 carboxylate.

A solid-phase assay for biotin was developed based on the difference in the dissociation constants of avidin—biotin (ca. 10 −15 M) and avidin—iminobiotin (ca. 10 −8 M) at pH 9.5. In this assay, avidin—peroxidase conjugates (AvP) were dissociated from AvP-immobilized iminobiotin complexes by the competitive binding of biotin, which has a higher affinity than iminobiotin for avidin. The extent of AvP dissociation which is dependent on the concentration of biotin, was determined by monitoring the activity of peroxidase enzyme conjugated to avidin. The beads could be reused following treatment with acetate buffer (pH 4.5) because the dissociation constant of iminobiotin is pH dependent. The detection limit for the assay was in the nanomolar range while the relative standard deviation for ten replicate assays of 0.1 μM biotin was 4.93% ( n = 10).

The preparation and characterization of rabbit and bovine IgG-coated membranes is discussed. The performance of these membranes in a competitive binding assay for rabbit IgG and in a sandwich assay for rabbit anti-bovine IgG were used to judge the potential usefulness of these membranes as solid phases for enzyme immunosensors. In particular, these membranes were intended for use in enzyme immunosensors based on the ammonia gas-sensing electrode which employed urease labeled immunoreagents.

Electrochemical detection of tricyclic antidepressants was conducted with glassy carbon, carbon paste and antimony doped tin oxide electrodes. We find that glassy carbon electrodes undergo passivation due to an adsorption process. Reliable calibration curves can only be obtained by polishing the electrode surface between scans. Carbon paste electrodes result in nonlinear calibration curves due to partitioning of the analyte and/or oxidized products of the analyte into the organic phase of the carbon paste. The extent of partitioning is dependent on the number of scans in the presence of the analyte. Antimony doped tin oxide yielded reproducible, linear calibration curves in the flow injection analysis mode as well as with differential pulse polarography.

The current status and future prospects for biosensors are viewed from a personal perspective of 30 years. It is suggested that the principal obstacles to progress in the biosensor field are at least as much human and organizational as scientific or technical.

Two species of Hawaiian crustaceans, Portunis sanguinolentus and Podophthalmis vigil, are shown to be effective for the construction of antennular receptrodes. The resulting chemoreceptor-based biosensors respond to stimulant compounds at levels below 10−15 M with very short response times. Experimental studies focused on trimethylamine oxide as a novel test compound, combined with chemometric techniques for data analysis, yield quantitative dose-response data over a wide concentration range.

Hydrogen peroxide was determined amperometrically in a steady-state arrangement by utilizing a kohlrabi - ferrocene based carbon paste electrode. A very short response time (2.6 seconds) and a relatively large usable pH range (5.0–7.4) were obtained. Several important hydrogen donors were studied as possible interferences.

The use of pea seedlings as a source of diamine oxidase for the determination of enzymatic oxidation rates, which are important in evaluating the plant growth-regulating activity, of various amines is reported. A mixed carbon paste-plant tissue amperometric sensor was constructed and characterized using speramidine as a substrate (KM=5 × 10−6 M). Generation of hydrogen peroxide due to enzymatic oxidation of amine was monitored at 0.9 V vs. Ag/AgCl under steady-state conditions using this sensor. Easy construction, reliability of the data, reusability, shorter response times (of the order of 4 s) and a detection limit of 7.1 nmol 1−1 are some of the advantages.

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.

A flow-injection analysis (FIA) system for the determination of hydrogen peroxide was developed by utilizing a pineapple-tissue-based modified carbon paste electrode. Pineapple stem core tissue contains peroxidase, which is not available commercially in the pure isolated form. Hydrogen peroxide concentrations were determined in the presence of O-phenylenediamine as a hydrogen donor at a potential of −200 mV. The detection limit (S/N = 3) for hydrogen peroxide using this tissue electrode is 2.1 μM with a relative standard deviation of 0.44% (n = 12). The tissue-modified carbon paste electrode is stable for up to 20 days with no significant activity changes.

A new receptor based assay is described for the determination of toxins which have high affinities for the acetylcholine receptor. The method is based upon the hindrance of the normal binding of a synthetic enzyme-drug conjugate with a high affinity for the acetylcholine receptor protein by the presence of toxins acting as antagonists. The activity of the enzyme marker system, glucose-6-phosphate dehydrogenase covalently conjugated to desipramine, is monitored by colorimetric detection of the rate for NADH formation at 340 nm. The procedure proposed is designed to provide a simple toxin screen which can be done in a minimally equipped laboratory while achieving the required sensitivity. The technique is illustrated for snake venoms from Bungarus multicintus, Naja naja, and the alkaloid tubocurarine. Aspecific binding responses are shown to have minimal effect on the assay.