Anatomy of a successful imprint: analysing the recognition mechanisms of a molecularly imprinted polymer for quercetin.

This study comprises a retrospective analysis of a successful molecular imprint for quercetin with the main aim of deriving rational design strategies for more effective molecularly imprinted polymers. Hence, polymers of varying composition were synthesised and chromatographically characterised to examine the effects of monomer-template ratios. (1)H NMR analysis of the pre-polymerisation mixture yielded further information on the nature of the complexes formed prior to the polymerisation step. A direct correlation between the optimum monomer-template ratio derived from the chromatographic studies and the monomer-template ratio providing the most stable pre-polymerisation complexes observed via (1)H NMR T(1) relaxation time measurements, suggests that the formation of particularly stable pre-polymerisation complexes is responsible for an increased formation of selective binding sites during the polymerisation step. Furthermore, physical aspects of the polymerisation, such as the MIP surface area and macroscopic phase partitioning of the mixture during the polymerisation are investigated. The observed effects and their analytical assessment offer insight into the mechanisms governing MIP selectivity at a molecular level.

Towards the rational development of molecularly imprinted polymers: 1H NMR studies on hydrophobicity and ion-pair interactions as driving forces for selectivity.

The preparation of molecularly imprinted polymers (MIP) based on non-covalent interactions has become a widely used technique for creating highly specific sorbent materials predominantly used in separation chemistry. A crucial factor in a successful imprinting protocol is the optimisation of the template/functional monomer interaction in the pre-polymerisation mixture, eventually leading to a maximum of high-affinity binding sites in the resulting polymer matrix. In order to develop more efficient preparation technologies for imprinted polymers, two separate pre-polymerisation complexes were investigated by NMR spectroscopic techniques in order to identify the types of interactions occurring in the pre-polymerisation mixture, and their implications for the subsequently formed imprinted polymer. In particular, hydrophobic effects have been followed by NMR spectroscopy and their contribution to the selectivity of the resulting MIP has been investigated. The 2,4-D imprint system is used as an example to fundamentally study whether observations at the pre-polymerisation stage correlate with properties of the finally prepared MIP, and which parameters govern success of an imprinting protocol.

Creatinine biosensors: principles and designs.

Creatinine biosensors, based on both potentiometric and amperometric devices, have been created. However, there are significant problems still to be addressed, including the balance between sensitivity and selectivity, interference rejection and sensor stability. In addition, many devices still rely on a dual-sensor approach for creatine and creatinine subtractive measurements. However, creatinine biosensors appear close to attaining the performance goals necessary for their widespread application. This article looks at the operating principle and design of both potentiometric and amperometric creatinine biosensors, and shows how the design of these devices affects their performance.

Bioavailability studies of oral dosage forms containing levodopa and carbidopa using column-switching chromatography followed by electrochemical detection.

A reliable multi-dimensional column chromatographic method employing amperometric detection using a carbon fibre microelectrode procedure was used for monitoring the plasma profiles and to evaluate the pharmacokinetics and bioavailability of levodopa (L-dopa) and carbidopa (C-dopa), after ingestion of oral formulations containing these drugs. The peak currents obtained for the different analytes were directly proportional to the analyte over the concentration range 0.02-4 micrograms ml-1. Using this method, the minimum detectable concentration was estimated to be 5 and 8 ng ml-1 for L-dopa and C-dopa, respectively. Recovery studies ranged from 93.83 to 89.76%, with a relative standard deviation of less than 7%. The study was carried out in two separate weeks on five healthy non-patient fasted male/female volunteers in the age range 20-37 years and weighing between 60 kg and 78 kg. The pharmacokinetic profile of two controlled-release products containing both L-dopa and C-dopa (Sinemet CR3 and CR4) was compared on the one hand and Sinemet conventional tablets on the other. The pharmacokinetic parameters, peak concentration (Cmax), the time taken to obtain this level (Tmax), elimination half-time T1/2, elimination rate constant (Kel), plasma level ratio, fluctuation index (FI) and the area under the time-concentration curve (AUC0-8), were investigated for each individual formulation. A comparison of the uptake of L-dopa from the conventional formulation showed that L-dopa entered the plasma and achieved peak levels higher than that of the controlled release formulations. However, it showed a much higher fluctuation index and the plasma concentrations were more stable with the controlled release formulations. The data also indicated a very low accumulation of both levodopa and carbidopa following repeated administration of the drugs, which was consistent with their relatively short half-lives (less than 2 h). In contrast, the half-life for the metabolite 3-orthomethyl dopa (3-OMD) is in the order of 13 h. As a result, there was an extensive accumulation of 3-OMD and its levels were significantly higher than those of levodopa or carbidopa upon repeated administration. Urine recoveries of the three analytes over one 8 h dosing interval showed that the majority of the excreted levodopa and carbidopa was recovered during the first 4 h, and there is proportionally greater excretion of the carbidopa dose than the levodopa dose.

Rapid antibody biosensor assays for environmental analysis.

Traditionally, biosensor development has focused on molecules with a defined metabolic role that can be exploited by enzyme-based systems. Antibodies have the ability to move beyond this range of analytes, and are particularly useful in detecting small, hapten molecules. Electrochemically based biosensor developments have been less fruitful in this regard, as enzyme labelling is required, and such assays require the separation from bound and unbound species. These separations and the removal of background signals result in the increased complexity of the assay format, making it unsuitable for rapid sensor analysis. We have developed an electrochemical sensor based on antibodies that does not require the separation of bound and unbound molecules in a competition immunoassay format. This removes the need for several washing and separation steps as is normally employed in this type of assay. This allows single-step immunoassays to be performed using this system, and also allows for the real-time monitoring of antibody-antigen interactions. We have shown that such assays are possible in both batch and flow-injection formats and we are currently developing an assay for the pesticide atrazine. Tentative results show that analysis with this system is possible in the p.p.m. to p.p.b. range.

Simultaneous determination of levodopa, carbidopa and their metabolites in human plasma and urine samples using LC-EC.

In this study levodopa (L-DOPA), carbidopa (C-DOPA) and their metabolites were resolved from other endogenous components present in human plasma and urine and determined quantitatively. The developed technique involved the use of a second pump, a switching valve, and a pre-column in the LC system in order to perform on-line sample clean-up and enrichment. This procedure is dependent on an effective removal of the many interfering matrix components that vitiate HPLC analysis. Several unknown endogenous electroactive compounds, present in plasma, were eliminated by the purification step, or suppressed by the pre-treatment or detection conditions. The analyses were separated on an Octyl-bonded reversed-phase column followed by amperometric detection using a carbon fibre microelectrode flow cell operated at +0.8 V versus silver/silver phosphate reference electrode. The cell was compatible with the mobile and the stationary phase used in the flow system without any complex surface reaction. The peak currents obtained for the different analytes were directly proportional to the analyse over the concentration range 0.02-4.0 microg ml(-1). Using this method, the minimum detectable concentration was estimated to be 5 and 8 ng ml(-1) for L-DOPA and C-DOPA, respectively. Recovery studies performed on human plasma samples ranged from 93.83 to 89.76%, with a relative standard deviation of < 6%. The intra- and inter-assay coefficients of variation were < 7%. The accuracy of the assay, which was defined as the percentage difference between the mean concentration found and the theoretical (true) concentration, was 12% or better. The electrochemical pre-treatment regime described in this work permitted a longer application of the same microelectrode. The method showed a good agreement with other available methods described in the introduction and offers the advantages of being simple, less time and labour consuming, does not require additional solvents for extraction, inexpensive and suitable for routine analysis and kinetic purposes.

A comparative bioavailability study of different aspirin formulations using on-line multidimensional chromatography.

A multi-dimensional column chromatographic method employing UV spectrometric detection was optimised and successfully used in a comparative bio-availability study of aspirin obtained from different commercially available oral dosage forms. Sample clean-up was achieved by on-line solid-phase extraction. In this study, the bioavailability of aspirin was compared in plain aspirin tablets, chewed tablets, effervescent tablets and Enteric-coated aspirin tablets. Blood samples were taken at frequent intervals after single dosing in ten healthy volunteers, the plasma samples were first treated with physostigmine sulphate to minimise enzymatic hydrolysis of aspirin to salicylate. The results showed the measured Tmax, Cmax, and AUC was significantly higher for soluble aspirin than for the other formulations and the t1/2 was shorter. This indicates the rapid absorption of aspirin from a soluble formulation compared with that from the other formulations. These differences suggest that the soluble formulation could be the aspirin of choice to treat patients suspected to be at high risk of myocardial infarction. The method performs, in a single step, an efficient extraction and clean-up of aspirin from human plasma. The calibration graph was linear over the calibration range 0.2-12 microg ml(-1) plasma with a limit of detection of 0.1 microg ml(-1). The intra- and inter-assay coefficients of variation were less than 6% and the recoveries ranged from 86 to 98%. The proposed method combines the advantages of being simple and selective in the presence of other potential interfering drugs and is suitable for routine analyses to obtain valuable information about the clinical effects of the drug and its use in prevention treatments of acute myocardial infarction. The whole procedure takes 7 min and is in agreement with other conventional methods.

Electrochemical detection of microcystins, cyanobacterial peptide hepatotoxins, following high-performance liquid chromatography.

A novel amperometric HPLC detection method for the cyanobacterial (blue-green algal) peptide toxins microcystin-LR, -YR and -RR was developed. Purified microcystins and cyanobacterial extracts were chromatographed using an internal surface reversed-phase column with acetate- and phosphate-based mobile phase systems. Electrochemical oxidation reactions at 1.20 V vs. Ag/AgCl (glassy carbon working electrode) were show to originate in arginine and tyrosine residues of microcystins.

Development of a capillary electrophoretic separation of an N-(substituted)-glycine-peptoid combinatorial mixture.

Capillary electrophoresis was used for the separation of a combinatorially synthesized N-(substituted)-glycine (NSG) peptoid mixture. This mixture consisted of 24 trimeric compounds sharing a common backbone structure but differing in the side chain attached at the N-terminal residue. Standards of the individual components were unavailable so that development of the separation was based on the mixture. A variety of buffer additives were investigated to enhance the CE resolution of this diverse mixture. Ion-pairing agents, cyclodextrins and organic modifiers were all evaluated as buffer additives. The best separations were achieved using a combination of buffer additives, each serving a different purpose in the separation. Heptane sulphonic acid (HSA) was used to reduce hydrophobic intramolecular interactions. Methyl-beta-cyclodextrin was used to provide host-guest interactions in order to resolve the very hydrophobic components of the NSG-peptoid mixture. The optimized run buffer consisted of 250 mM sodium phosphate buffer, pH 2.0, with 25 mM HSA and 40 mg/ml BCD and resulted in the resolution of 21 peaks for the 24 peptoids in the combinatorial mixture.

Application of capillary electrophoresis to the separation of structurally diverse N-(substituted)-glycine-peptoid combinatorial mixtures.

The capillary electrophoresis (CE)-based separation of five N-(substituted)-glycine (NSG)-peptoid mixtures with a wide range of physical and chemical properties was studied. A CE separation, initially developed using a single representative peptoid mixture, with a background electrolyte (BGE) modified by the addition of both methyl-beta-cyclodextrin and heptane sulfonic acid was found to provide good separations of most of the combinatorial mixtures investigated. For those mixtures not separated well by this procedure, the use of SDS micelles in conjunction with methyl-beta-cyclodextrin resulted in dramatic improvements in the separation. While no single set of separation conditions proved sufficient for all of the NSG-peptoid combinatorial mixtures, the two methods were able to provide separation sufficient for characterization of a set of mixtures with a wide range of physical and chemical properties. The efficiency of the CE-based separation of the combinatorial mixtures studied was compared to a reversed-phase liquid chromatographic method using gradient elution.

Drug metabolism biosensors: electrochemical reactivities of cytochrome P450cam immobilised in synthetic vesicular systems.

Biosensors containing cytochrome P450cam in a didodecyldimethylammonium bromide vesicular system were prepared by cross-linking onto a glassy carbon electrode (GCE) with glutaraldehyde in the presence of bovine serum albumin. Cyclic voltammetric responses of the sensor in air-free buffer solution showed that the sensor exhibited reversible electrochemistry due to direct electron exchange between the haem Fe(3+/2+) redox system and the GCE surface. In air-saturated solution containing camphor, the biosensor gave an irreversible electrocatalytic current which is compatible with the monooxygenation of the substrate. Steady state amperometric experiments with camphor, adamantanone and fenchone were performed with a biosensor prepared by cross-linking P450cam with glutaraldehyde onto a Pt disc electrode. The sensor was characterised by fast amperometric responses, attaining steady-state in about 20 s in a cobalt sepulchrate mediated electrochemical system. The kinetic parameters of the biosensor were analysed using the electrochemical Michaelis Menten equation. The estimated apparent Michaelis-Menten constant, Km, values for the biosensors were in the range of 1.41-3.9 mM.

Multi-residue analysis of penicillin residues in porcine tissue using matrix solid phase dispersion.

The aim of this study was to develop a multi-residue method for the analysis of penicillins in animal tissue. Matrix solid phase dispersion (MSPD) was employed to extract the residues and the extracts were then cleaned-up by C18 solid phase extraction (SPE). Pre-column derivatisation using acetic anhydride and 1,2,4-triazole in the presence of mercuric chloride was employed to allow detection in 325 nm. Gradient elution was required to elute amoxicillin, ampicillin, penicillin G, cloxacillin and dicloxacillin derivatives from a C18 reversed phase column using phosphate buffer-acetonitrile mobile phase. The developed method had a limit of detection of 20 ng g-1 and had recoveries in the range 40-90% for the 5 drugs in samples fortified at 40 and 200 ng g-1; the maximum residue limits (MRLs) for these drugs were in the range of 50-300 ng g-1 (ppb).

The preparation of a molecular imprinted polymer to 7-hydroxycoumarin and its use as a solid-phase extraction material.

A molecular imprinted polymer (MIP) was prepared to 7-hydroxycoumarin (7-OHC). A number of preparation parameters were examined by ultraviolet (UV) spectroscopy, including the amount of solvent used for reaction, equilibration time, selectivity and capacity of material. The polymer which showed the most selectivity for 7-OHC was then packed into cartridges and used as a solid-phase extraction sorbent. An extraction procedure was then developed from first principles. The cartridges were examined for selectivity of 7-OHC over some other members of the coumarin family. 7-OHC was then extracted from urine using this solid-phase extraction (SPE) method, and its concentration determined using capillary zone electrophoresis (CZE). The method was found to be linear over the range 10-50 micrograms ml-1. Inter- and Intra-assay precision studies were performed to validate the method.

Voltammetric detection for capillary electrophoresis.

Several approaches to implementing amperometric detection for capillary electrophoresis have been reported. This report describes the development of a voltammetric detector for CE. The detector is designed to minimize distortion of the voltammetry due to ohmic potential drop. This was accomplished by using a cast Nafion detection cell at the end of the separation capillary. The cast Nafion detection cell provided a low-dead-volume, low-resistance cell that minimized ohmic potential drop and peak band broadening. The ability to detect the current due to oxidation of analytes superimposed on a large background current was also improved. A dynamic background subtraction scheme was used in which a second working electrode, positioned in the electrochemical cell but outside of the detection cell, was used to compensate for the background current in real time. The output of the compensating working electrode was subtracted from the output of the detecting working electrode prior to analog-to-digital conversion. Postexperimental digital background subtraction was also implemented. This approach provided optimal elimination of the background current with maximal detection of the analytical signal. The voltammetric detector developed produced high-quality voltammetric response of analytes with injected concentrations as low as 0.20 microM. The system was evaluated by obtaining CE voltammograms of a mixture of eight test phenolic acids.

Sol-gel based amperometric biosensor incorporating an osmium redox polymer as mediator for detection of L-lactate.

A novel amperometric biosensor for the determination of lactate was constructed by first immobilizing lactate oxidase and an osmium redox polymer ([Os(bpy)(2)(PVP)(10)Cl]Cl; abbreviated Os-polymer) on the surface of a glassy carbon electrode, followed by coating with a sol-gel film derived from methyltriethoxysilane (MTEOS). The electrooxidation current of this electrode was found to be diffusion controlled. In the presence of lactate, a clear electrocatalytic oxidation wave was observed, and lactate could be determined amperometrically at 400 mV versus Ag AgCl . The concentration range of linear response, slope of linear response and detection limit were 0.1-9 mM, 1.02 microA mM(-1), and 0.05 mM, respectively. Although L-ascorbate was electrooxidized at this potential, uric acid, paracetamol and glucose were found not to interfere.

In vitro optimisation of a microdialysis system with potential for on-line monitoring of lactate and glucose in biological samples.

The optimisation and evaluation of the microdialysis component of a prototype miniaturised total analysis system for application in the continuous monitoring of lactate and glucose is reported. The complete unit comprises a high efficiency microdialysis sampling system, a miniaturised microflow manifold with an integrated biosensor array, together with the hardware and software necessary for controlling the flow parameters and monitoring the sensor signals. Sampling occurs via a microdialysis shunt probe which is perfused continuously with a physiological buffered saline solution. The continuous dialysate outflow is presented to the biosensor array, resulting in the appropriate amperometric signals. Aspects of technological significance addressed here include probe membrane size, perfusate flow rate, sample flow rate, temperature change, probe sterilisation procedures, and heparin content of the physiological saline solution employed.

Effects of acetonitrile on horseradish peroxidase (HRP)-anti HRP antibody interaction.

The effects of the water-miscible organic solvent acetonitrile on the enzymatic activity of horseradish peroxidase (HRP) and on HRP-anti-HRP binding have been investigated. Results showed that both the catalytic activity of HRP and the binding ability of the antibody were affected on increasing the concentration of the organic solvent. The activity of HRP varied with the organic composition of the solvent, indicating that the conformation of the enzyme was affected. The binding ability of the antibody also decreased significantly with an increase of the organic composition of the solvent, and in absolute acetonitrile, the activity of the antibody is about 500 times lower than that in aqueous medium. Binding reversibility experiments indicated that the antibody was not irreversibly damaged in solutions with acetonitrile composition greater than 80% and below 40%; however, an irreversible decrease in the binding was observed in solutions with an acetonitrile composition between 40 and 80%. The reduction in the binding ability is probably due to the irreversible conformation changes in the antibody.

Sol-gel carbon composite electrode as an amperometric detector for liquid chromatography.

The performance characteristics of an electrochemical detector for liquid chromatography based on a sol-gel carbon composite working electrode in a wall-jet configuration are described. The new detector combines the versatility of sol-gel processes with several favorable characteristics, including fast electron-transfer kinetics, mechanical rigidity and renewability. Factors influencing the amperometric response are explored and optimized. Detection limits of 58-170 pg are reported for various neurotransmitters. Repetitive injections yield peak heights with relative standard deviations of 2.6-3.7%. The prospects of using sol-gel derived electrochemical detectors are discussed.

Screen-printed tyrosinase-containing electrodes for the biosensing of enzyme inhibitors.

Disposable amperometric inhibition biosensors have been microfabricated by screen printing a tyrosinase-containing carbon ink. The decrease in the substrate (catechol) steady-state current, caused by the addition of various pesticides and herbicides, offers convenient quantitation of micromolar levels of these pollutants. Unlike esterasebased disposable strips, the tyrosinase thick-film devices can be fabricated by incorporating the enzyme within the carbon ink. and do not require a prolonged incubation step in the presence of the inhibitor. The effect of experimental variables, such as the enzyme loading or substrate concentration, is assessed. Applicability to an untreated river water sample is illustrated. Such use of single-use devices for monitoring toxins addresses the problem of irreversible enzyme inhibition, and holds great promise for on-site field analysis.

Differential-pulse voltammetric determination of clenbuterol in bovine urine using a Nafion-modified carbon paste electrode.

A method has been developed for the determination of clenbuterol in bovine urine using differential-pulse voltammetry (DPV), based on the electrochemical behaviour of clenbuterol at a Nafion-modified carbon paste electrode (CPE). Clenbuterol is irreversibly oxidized at high positive potentials, its irreversibility being due to a chemical follow-up reaction which results in a product showing quasi-reversible electrochemical behaviour at much lower potentials. It is the oxidation peak of this product, arising in acidic media at 0.42 V, which was analysed using DPV, again following the accumulation of clenbuterol at the Nafion-modified CPE. Electrode renewal was achieved by holding the potential at -0.6 V for 120 s in 0.1 mol l-1 NaOH. The determination of clenbuterol in the presence of interfering compounds present in bovine urine samples was then carried out after a two-step clean-up of the urine involving liquid-liquid extraction followed by a mixed-mode solid-phase extraction procedure. This allowed clenbuterol to be detected down to a level of 1.02 x 10(-9) mol l-1 in bovine urine extracts.