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Biological molecules (e.g., antibodies, enzymes and receptors) have been widely used as specific recognition elements in analytical assays, from homogeneous assays to biosensors, for applications in healthcare, environmental monitoring and industrial-process control. Limited stability, and difficulty and high cost of production are their main drawbacks. Artificial receptors and catalysts prepared by molecular imprinting technology are valuable in replacing biomolecules for molecular recognition in these kinds of assay. In this review, we describe the most recent developments in non-covalent molecularly-imprinted polymers (MIPs) to replace natural receptors in ligand-binding assays and biosensors. A key factor underpinning the most significant advances in this field is obtaining materials that improve the specific recognition and, at the same time, favor transduction. Composites of MIPs and carbon nanostructures or inorganic nanoparticles combine the recognition properties of MIPs with the electrical, optical or magnetic properties of the nanomaterials, resulting in new imprinted nanoreceptors with great potential as sensing materials. We summarize various methods to blend MIPs with different nanomaterials and highlight their application in biomimetic assays. Electropolymerized MIPs (E-MIPs) offer an option for integrating the recognition elements as nanofilms on the surface of different transducers. We discuss recent advances in the use of E-MIPs for analytical applications. MIPs mimicking natural enzymes are also among these new functional materials. We present the approaches to prepare catalytic non-covalent MIPs along with recent analytical applications and challenges for future developments.

Acrylic polymers with catalytic activity for the oxidative degradation of 2,4,6-trichlorophenol (TCP) were developed. In order to mimic the active site of chloroperoxidase (CPO), chloro-iron(III)-protoporphyrin IX was used as the catalytic centre, and methacrylamide (MA) and 4-vinylpyridine (VPY) were used as the monomers that build up the active sites. Taking as basis 3:1 (w/w) acid:basic aminoacidic composition of CPO, three MA:VPY combinations were tested: one keeping the same ratio (3:1) i.e. 25% VPY in the functional monomer mixture, one with lower content of the basic monomer (9:1) i.e. 10% VPY, and one with higher concentration of it (1:1) i.e. 50% VPY. Polymers synthesized with the lowest VPY content exhibited the highest catalytic efficiency, which was improved by the creation of specific TCP binding sites through molecular imprinting technology. In these way, synthetic enzymes with useful properties for analytical and bioremediation applications were obtained.

A method for the rational design of molecularly imprinted polymers with catalytic activity (MICs) that mimic hemeproteins is described. Density Functional Theory calculations applied to the pre-polymerization adduct and to a possible reaction intermediate allow to choose the best functional monomer that builds up the active sites. This functional monomer should stabilize both relevant stages, the first one in an adequate porogenic solvent and the second one in the aqueous reaction medium. In this work, a MIC was synthesized to catalyze the oxidation of 2,4,6-trichlorophenol (TCP), the substrate and template molecule. A hemin molecule was used as the catalytic centre and four functional monomers and six solvents were tested. According to the theoretical predictions, methacrylamide and 4-vinylpyridine MIPs successfully mimic the recently discovered ability of Chloroperoxidase (CPO) to catalyze the oxidative dechlorination of TCP in batch analysis. They exhibit good catalytic properties and substrate selectivity.

An RNA aptamer is proposed as a recognition element for the detection of tobramycin in human serum. A displacement assay was developed using faradaic-electrochemical impedance spectroscopy (F-EIS) as a detection technique. Two modified aptamers, a partially (ATA) and a fully O-methylated aptamer (FATA) were evaluated and compared. The affinity constant, K(D), for both aptamers was estimated by F-EIS resulting virtually identical within the experimental error. The selectivity towards other aminoglycosides was also studied. The analytical characteristics were evaluated in aqueous solution using both aptamers and FATA was selected for human serum experiments. Using a 1:0.5 dilution of the serum, a linear range between 3 μM and 72.1 μM was obtained, which included the therapeutic range of the antibiotic.

Gluten analysis in food is intricate because of the analyte variability among cereal species and protein states. Herein the immunodominant peptide 33-mer from alpha2-gliadin is proposed as standard for calibration in a competitive ELISA assay with amperometric flow-injection detection. Using the monoclonal antibody G12, a correlation between the content of gluten in peptide and gluten units was obtained for the first time. The analytical features were compared with optical measurements using several standards obtaining a slightly lower detection limit, which suggested the sensitivity is limited by affinity and not by the detection technique. “Gluten-free” samples were satisfactorily analyzed.

The detection of small molecules using aptamers is not straightforward when the induced fit triggered by the analyte is also small. Herein a labeling strategy is explored for the voltammetric detection of the antibiotic tobramycin using magnetic microparticles (MPs). A competition between MPs-immobilized tobramycin and free tobramycin for the limited amount of the biotinylated-aptamer was established. The addition of the enzymatic conjugate and the substrate generated a product that was electrochemically oxidized on screen-printed carbon electrodes. The analytical signal was proportional to the antibiotic concentration from 5–500 µM and the reproducibility was 5.7 %. The assay selectivity was also studied.

This article describes the synthesis and characterization of a catalytic 2,4,6-trichlorophenol molecularly imprinted microgel that could replace a natural chloroperoxidase to develop electrochemical sensors. This polymer exhibits an apparent mean particle size of 1–4 µm and high porosity, and mimics the dehalogenative function of the natural enzyme chloroperoxidase for p-halophenols. Two strategies were explored: a carbon paste modified with the polymer and the drop-coating of screen-printed electrodes with powder suspensions of the polymer and carbon nanotubes. With this last design, TCP concentrations above 25 µM could be detected. The sensor developed this way could be used for environmental waste screening.

A sigmoidal behaviour of chloroperoxidase for the oxidative dehalogenation of 2,4,6-trichlorophenol is reported for the first time. Kinetic data were adjusted to the Hill equation and the kinetic parameters were obtained: n = 1.7 ± 0.2, v max = ( 8.8 ± 0.3 ) × 10 − 5 M mi n − 1 , the pseudo-Michaelis constant Ks* = (8.6 ± 0.5) × 10−5 M, kcat = 677 ± 84 min−1 and the catalytic efficiency = (8.9 ± 0.6) × 106 M−1 min−1. The sigmoidal curve could be related to the cooperative binding of the substrate to the enzyme, so that the binding of the first substrate molecule may help the binding of the second one. Further, both substrate molecules could establish Π–Π interactions between them, which would confer more stability to the system.

A comparative evaluation of an electrochemical sandwich genoassay for pathogenic bacteria based on immobilized hairpin DNA probes and three different enzyme labels (horseradish peroxidase, alkaline phosphatase and a biomimetic peroxidase-like DNAzyme) is reported. The natural enzymes were used as streptavidin conjugates, coupled to the surface duplex by using a biotin-labeled signaling probe, whereas the DNAzyme was directly incorporated to the sequence of the signaling probe. HRP provides enhanced sensitivity although the choice of a catalytic reporter DNA sequence could simplify the assay.

The synthesis, characterization and evaluation of the catalytic properties of a hemin–methacrylamide–ethylene glycol dimethacrylate copolymer are described. This polymer catalyzes the oxidative dechlorination of 2,4,6-trichlorophenol (TCP) in the presence of hydrogen peroxide, yielding 2,6-dichloro-1,4-benzoquinone as the main reaction product. This low-cost material allows the complete removal of TCP from water at concentration level of 20 mg L −1 in less than 30 min.

The use of nucleic acid with a specific sequence and a highly ordered secondary structure such as hairpins, quadruplexes and pseudoknots as biological recognition elements and switches in biosensors is rapidly increasing because of their improved features (e.g. selectivity) when compared with the traditional linear probes. Owing to the novelty, a critical outlook of their characteristics and a compilation of the latest advances are lacking. This article describes the potential of those nucleic acids probes whose molecular recognition ability relies on a conformational change (e.g. folding/unfolding mechanism) in electrochemical sensing. It provides an overview of the toolbox of assays using these probes for genosensors and aptasensors, highlighting its performance characteristics and the prospects and challenges for biosensor design.

A carbon paste-poly(o-phenylendiamine)-modified electrode to be used as amperometric biosensor for 2,4,6-trichlorophenol (TCP) is described. The enzyme chloroperoxidase (EC 1.11.1.10) from Caldariomyces fumago is immobilized through dispersion in a graphite paraffin oil carbon paste covered by an electrogenerated poly(o-phenylendiamine) (PPD) layer. The main enzymatic dehalogenation product, 2,6-dichloro-1,4-benzoquinone (DCQ) is characterized by liquid chromatography-mass spectrometry and cyclic voltammetry. This product is electrochemically active and can be detected amperometrically at +150 mV vs. Ag|AgCl|KCl (3 M). The biosensor exhibits a response time of 4 min, a detection limit of 10−7 M, and a dynamic linear range between 10−7 and 10−6 M. Selectivity as well as operating and storage stability were evaluated.

We have studied how the modification of the RNA aptamer evolved against neomycin B at 2' position of ribose with a methyl group influences the affinity of the interaction. Using surface plasmon resonance (SPR) and faradaic impedance spectroscopy (FIS) an affinity constant in the muM range was calculated. The results showed that the modification of the aptamer does not significantly alter the affinity of the aptamer for the antibiotic. This finding opens up the possibility of designing modified RNA aptamers resistant to endonucleases without variation of the analytical features. In addition to this, we propose a competitive assay for the detection of neomycin B using SPR as a transduction technique. A range of quantification between 10 nM and 100 microM was obtained, which shows the feasibility of detecting small molecules using aptamers with high sensitivity.

Human infections with Legionella pneumophila represent a public health problem. Current culture assays for surveillance and control of L. pneumophila in water are time-consuming and limited by the sensitivity, especially when samples also contain microorganisms that inhibit Legionella growth. In this work, an electrochemical method, different from real-time polymerase chain reaction (PCR) approaches, for semiquantitative evaluation of L. pneumophila is presented. A PCR assay targeting the 16S-rRNA gene of L. pneumophila giving rise to a 95-mer amplicon was established. Amplicons were hybridized to a biotin-labeled reporter sequence and then to a thiolated stem-loop structure immobilized onto gold electrodes as a reporter molecule with 1-naphthyl phosphate as a substrate. 1-Naphthol enzymatically generated was determined by differential pulse voltammetry (DPV). For a constant number of amplification cycles, results show that the voltammetric signal is related to the number of copies in the sample thus achieving a useful semiquantitative estimation of L. pneumophila. After 40 cycles of PCR amplification this methodology has a limit of detection of 10 genomes, allowing the reliable detection of 10(2) genomes of L. pneumophila as well as distinguishing 10(3) and 10(4) genomes of the pathogen, values related to corrective actions in water systems in buildings, in accordance with the legislation currently in force.

A disposable voltammetric sensor for the detection of DNA sequences specific of Legionella pneumophila is presented. The sensor is based on a sandwich-hybridization format assay, using a stem-loop DNA structure as capture probe immobilized onto a screen-printed gold electrode. The signaling-DNA probe is labeled with biotin, and streptavidin-alkaline phosphatase is used as reporter molecule. 1-Naphthol, the enzymatic hydrolysis product of anaphthyl phosphate, is voltammetrically detected. The hairpin-modified screen-printed gold electrode allows the detection of a 52-mer DNA sequence at 400 pM level. With a sample volume of 40 mL, this concentration corresponds to ca. 16 fmol of a DNA sequence specific of L. pneumophila. The analytical performance of this platform is compared with that previously obtained for a sensor constructed on a conventional disk gold electrode with the same geometry (˘ ¼ 1.6 mm) and design. A lower quantification limit is obtained with the SPE. In addition, the selectivity is improved as the disposable device shows a better discrimination between L. pneumophila and L. longbeachae or L. micdadei under identical nonstringent conditions.

The electrochemical oxidation of guanosine and xanthosine, which only differ in the presence of an oxygen atom at C2 position instead of an amine group, is studied on pyrolytic graphite electrodes at different electrolysis time scales in solutions of physiological pH. The oxidation products adsorbed on the electrode surface were electrochemical characterized in terms of variation of the formal potential with pH and catalytic activity towards the oxidation of NADH. The results were compared with those previously described for the oxidation of guanosine and adenosine in alkaline solutions. A common oxidation product is described, the oxidized form of 9-β-D-ribofuranosyluric acid, which is proposed as the point of convergence in the oxidation routes of all three purine nucleosides.

We have studied the potential electrocatalytic activity towards the oxidation of NADH of several oxidation products of guanine and its derivative guanosine-5′-monophosphate (5′-GMP) on pyrolytic graphite electrodes (PGE). The distribution of products generated strongly depends on the experimental conditions. Our investigations focused on the oxidation products that are adsorbed on the electrode surface, are redox active and, exhibited electrocatalytic activity toward the oxidation of NADH. These compounds were electrochemically and kinetically characterized in terms of dependence of the formal potential on pH and electron transfer rate constant ( k s ). The voltammetric and catalytic behavior of both guanine and 5′-GMP oxidation products was compared with that of other guanine derivatives we have previously studied. Some mechanistic aspects concerning the generation of the catalysts are also discussed.

A molecularly imprinted polymer for diclofenac (DCF) was prepared by thermal polymerization over silica beads using 2-(dimethylamino)ethyl-methacrylate as functional monomer. After silica elimination by HF treatment, the polymer was applied to the selective solid-phase extraction of the drug from urine followed by its quantification by adsorptive differential pulse voltammetry. Results indicate that the drug could be selectively extracted from the sample and quantified at clinically relevant concentrations (μg/mL).

We herein explore a novel route for oxidative stress in DNA by using electrochemistry as mimicking tool. Essentially, the electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes in neutral and alkaline aqueous solutions was studied. Under these experimental conditions all these compounds give rise to an adsorbed product not previously described, which was electrochemically and kinetically characterized. The virtually identical kinetic and electrochemical features exhibited by the oxidized compounds generated from all precursors strongly suggest a common species arising from both adenine and guanine derivatives. Supported by DFT calculations, we propose two convergent pathways for the electrochemical oxidation of adenosine and guanosine. Those calculations indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine. Keywords: Cyclic voltammetry, Density functional calculations, Electrochemistry, Guanosine, Substitution mutation

Auger electron spectroscopy (AES), linear sweep voltammetry and coulometry have been used to investigate the effect of bismuth surface coverage over the electron-transfer kinetics of the quinone/hydroquinone (Q(aq)/H2Q(aq)) redox couple at Bi-coated polycrystalline platinum (Ptpoly) electrodes. In 0.5 M HClO4 acid solutions, the observed rate of electron-transfer is controlled by the relative bismuth surface coverage present at the Ptpoly electrode surface. At full bismuth monolayer coverages, the reversibility of the Q(aq)/H2Q(aq) redox couple is considerably enhanced. Continued potentiodynamic cycling of the Q(aq)/H2Q(aq) couple at a Bi-coated Ptpoly electrode leads to a decrease in the Q(aq)/H2Q(aq) redox rates. The decrease in redox rates is accompanied by the simultaneous decrease in Bi(ad) surface coverage and a concomitant increase in the chemisorption of hydroquinone-derived adspecies. Eventually, all Bi(ad) is displaced and electron-transfer takes place slowly, through a spontaneously adsorbed hydroquinone-derived adlayer. In sharp contrast, in the presence of 1 mM Bi ( aq ) 3 + , no changes are observed during potentiodynamic cycling of the Q(aq)/H2Q(aq) redox couple and the reaction rates remain fast.

The voltammetric characteristics of catecholamines: epinephrine (E) and norepinephrine (NE) and related compounds: isoproterenol, metanephrine, L-dopa, methyldopa, vanillylmandelic acid (VMA), and homovanillic acid (HVA) at unmodified and in situ surfactant- modified carbon paste electrodes were comparatively evaluated. For the basic and amphoteric compounds the modification of the electrode surface with submicellar concentrations of anionic surfactants (sodium dodecylsulfate, sodium decylsulfate or sodium dodecylsulfonate) produce an important current enhancement in its oxidation and reduction peak current together with the improvement in the reversibility of the processes. These effects were explained in basis on electrostatic and hydrophobic interactions. On the other hand, the oxidation of acidic metabolites, HVA and VMA, was studied at electrodes modified in situ with cationic surfactants. Under certain conditions the surfactant could stabilise some of the electrochemical reaction intermediates, thus explaining the different voltammetric behaviour of HVA and VMA.

A simple and sensitive adsorptive- differential pulse- voltammetric method for the determination of flufenamic acid (FFA) is presented. The method is based on the preconcentration of FFA on carbon paste electrodes modified in situ with cationic surfactants, dodecyltrimethylammonium chloride (DTAC) or cetyltrimethylammonium chloride (CTAC), at submicellar concentrations. The best results were obtained with DTAC-modified electrodes. After optimization, with these electrodes and using an accumulation step at 0 V for 3 minutes in 0.1 M phosphate solutions pH 7, the peak current of the main oxidation process of FFA varies linearly with its concentration from 1×10−9 M to 5×10−5 M, with a limit of detection of 0.64 nM (0.2 ng/mL). The proposed method has been applied to the determination of FFA in spiked serum samples, treated with acetonitrile to separate the proteins. In the concentration range studied (3 μM to 65 μM) the recovery was near 100 % and the lowest concentration attainable in these samples is below 3 μM (0.8 ppm).

The generation of a new electrocatalytic system for NADH after oxidizing flavin adenine dinucleotide (FAD) is shown. The oxidation is performed in alkaline medium until +1.4 V (Ag/AgCl) at graphite electrodes. The catalytic activity is ascribed to the electrooxidized moiety of FAD and not to quinone surface groups. A comparison between this catalyst and that attributed to poly(FAD) (Karyakin, A. A.; Ivanova Y. N.; Revunova, K. V.; Karyakina, E. E. Anal. Chem. 2004, 76, 2004-2009.) is presented. It is concluded that the surface quinone groups generated during the strong anodization of the electrode in acidic medium at 2-2.5 V and not the poly(FAD) are responsible for the catalytic activity described in the above mentioned work.

Redox properties of two coenzymes, flavin adenine dinucleotide (FAD) and coenzyme A (CoA) are studied on pyrolytic graphite electrodes. Both of them exhibit an oxidation process at about 1.2 V in pH 9, associated with the oxidation of their adenine moiety. The resulting adsorbed oxidation products have a common structure, an electroactive quinone-imine that acts as efficient catalyst of the oxidation of NADH at low potentials. The influence of the structure on their electrochemical features in comparison with other adenine derivatives is studied. Finally, a modified electrode prepared with the oxidation product of CoA was used to determine NADH in a wide linear range (1×10−8–2.43×10−5 M) with a detection limit of 3 nM.

A new and simple electrochemical method capable to detect single-base mutations in DNA has been developed. It relies on the electrocatalytic oxidation of NADH by the intercalator Brilliant Cresyl Blue (BCB). Whereas negligible catalytic responses were obtained with graphite electrodes modified by BCB-single-stranded DNA, high catalytic activity is obtained at electrodes modified by the complexes between double-stranded DNA and BCB. The proposed explanation for these results is that charge transport from the intercalated BCB through the DNA base stack is a necessary event for the electrocatalytic activity as disruption by a single-base mismatch leads to a complete suppression of the electrocatalytic voltammetric response. These finds open new possibilities for the detection of DNA hybridization and mutation.

The electrochemical behavior of the antibiotics rifampicin and rifamycin SV is investigated by cyclic voltammetry at carbon paste and in situ surfactant modified carbon paste electrodes. Both antibiotics adsorb on the unmodified electrodes and show a reversible redox process due to the oxidation of the 6,9-dihydroxynaphthalene moiety to the corresponding naphthoquinone. This process is used as analytical signal for developing adsorptive voltammetric methods for the determination of the antibiotics. Experimental parameters, such as pH of the supporting electrolyte, accumulation potential and time are optimized. After accumulation from acidic solutions (0.1 M KCl pH 2 or HCl 0.2 M) at −0.1 or 0 V for 3 min, the differential pulse oxidation peak current changes linearly with the antibiotic concentration in the range 3.5×10−10 M −5.4×10−9 M or 5×10−11 M −1.0×10−9 M for rifampicin and rifamycin SV, respectively. Rifamycin SV is not accumulated on carbon paste electrodes modified in situ with the anionic surfactant sodium dodecyl sulfate, whereas rifampicin is readily accumulated on this modified electrodes resulting in a signal enhancement and allowing rifampicin determinations without interference from rifamycin SV. On the other hand, selective determination of rifamycin SV in the presence of rifampicin is achieved by using carbon paste electrodes in situ modified with the cationic surfactant cetyltrimethylammonium chloride.

The knowledge of the redox chemistry of nucleic acids (NA) is of paramount importance in cancer and aging research. Charge migration through DNA is also involved in biologically relevant functions such as DNA damage and repair. In the first part of this article the main aspects of the electrochemistry of nucleic acids at solid electrodes are revised, including redox processes, photoelectroactivity and electrical conductivity. In the second part, an overview of its applications is presented. Methods for electrochemical detection of NA, NA-based biosensors for detection of nonnucleic acid molecules, studies on the nature and dynamics of interactions and structural conformations of NA, are some applications that take advantage of NA electrochemistry at solid electrodes.

s-Adenosyl-L-methionine (SAMe) is an adenosine analogue with therapeutical activity against affective disorders and liver dysfunctions. It can be oxidized on graphite electrode yielding a strongly adsorbed electroactive oxidation product for which a quinone-imine structure is proposed. This compound is capable of electrocatalyzing the NADH oxidation at low potentials, lowering the overvoltage by about 300 mV. An amperometric method for NADH determination at +0.1 V (Ag|AgCl|KClsat) is developed using an oxidized-SAMe-modified electrode in pH 9. Linear calibration plots were obtained with a detection limit of 2.4 nM. The electrode response time and the relative standard deviation of the slope of the calibration plot for 5 different modified electrodes were 12 s and 5.6% respectively. The catalytic scheme also provides the first method to determine SAMe itself by adsorptive differential pulse voltammetry. The linear range was found to be 42.4–424 nM with a reproducibility of 6.9%. The method was applied to SAMe determination in a pharmaceutical formulation.

This article summarises our work on the development of voltammetric sensors based on molecularly imprinted polymers. Several recognition elements and integration strategies were used:1.membranes electropolymerised at the electrode surface; 2.casting of polymeric membranes by drop-coating a solution of pre-formed polymer (polyphosphazene) and template in a low-boiling-point solvent on to the electrode surface; 3.preparation of composite membranes containing conductive material (graphite or carbon black), acrylic-type molecularly imprinted polymers (small particle size), and PVC as binder; and 4.in-situ polymerisation of a thin layer of acrylic imprinted polymer deposited on the electrode surface by spin coating. All the options evaluated offer the possibility of controlling electrode characteristics such as hydrophobic/hydrophilic character, permeability, or film thickness, which are essential for obtaining good sensor performance.

Fludarabine (F-Ara-A, 9-β- d -arabinofuranosyl-2-fluoroadenine) is an arabinosyl nucleoside with potent cytotoxic activity against chronic lymphocytic leukemia. Herein, its electrochemical behavior is investigated on pyrolytic graphite electrodes (PGEs); and a novel catalytic adsorptive differential-pulse-voltammetric method for its determination is developed based on the strong electrocatalytic effect on nicotinamide adenine dinucleotide (NADH) oxidation exhibited by the oxidation products of F-Ara-A. This approach was applied to human urine samples, which required a previous solid-phase extraction. The F-Ara-A recovery was of 89.7%. The method proposed is fast, simple and permits detection of F-Ara-A concentrations up to 72 h after infusion.

A review of current strategies aimed at detecting nucleic acids (NA) using NA-modified solid electrodes reveals the versatility and potential of electrochemical detection in this field. What emerged at the beginning of 90s as a very promising detection system in DNA technology is now resulting in the first commercial devices. Many aspects of the experimental design, for example surface immobilisation and detection schemes, are outlined and evaluated. Although most approaches use hybridisation as the recognition reaction, those not based on hybridisation are also included. As is finally shown, great advances have been achieved, although further developments are required if electrochemical devices are to be suitable for routine measurement.

The electrochemical behavior of diclofenac (DCF) at graphite electrodes was investigated. The drug is irreversibly oxidized at 0.84 V (pH 1), giving rise to some products, which exhibit two reversible process with formal potential of 0.39 and 0.63 V. Carbon composite electrodes of different nature [polytetrafluoroethylene‐graphite (PTFE‐G), epoxy‐graphite (EG), epoxy‐carbon black (E‐CB)] were also investigated for voltammetric detection of DCF. A similar electrochemical behavior was observed on these electrodes although graphite was preferred over carbon black as conducting phase, and both PTFE and epoxy could act as agglomerant. Electrodes constructed with these materials could be modified with molecularly imprinted polymers (MIPs), and allowed detection of DCF after incubation in an acetonitrile solution of the drug.

Molecularly imprinted polymers (MIPs) are becoming an important class of synthetic materials mimicking molecular recognition by natural receptors. This review examines the literature on non-covalent MIP-based electrochemical sensors over the last 10 years. With insight into the different sensing phases, electrochemical transductions and integration strategies, we evaluate achievements and difficulties to date and assess future prospects.

A voltammetric sensor for the determination of diclofenac was developed, based on the molecular recognition of the analyte by molecularly imprinted methacrylate-ethyleneglycol dimethacrylate co-polymers. Pre-polymerisation solutions were deposited onto the surface of a glassy carbon electrode and a polymer film was obtained after spin coating control of thickness and in situ thermal polymerisation. After the template extraction from the resultant film, re-binding of diclofenac is performed from acetonitrile solutions containing the analyte. The amount of bonded diclofenac was then evaluated by differential pulse voltammetry in different electrolytes. The best results were obtained in 0.025 M citrate solution pH 6 containing 10% of acetonitrile. This medium favours the release of diclofenac from the polymer binding sites. In this way, the voltammetric transduction of the molecular recognition event is achieved. Voltammetric selectivity measurements revealed negligible interferences from diclofenac family of anti-inflammatory drugs, such as niflumic or meclofenamic acids.

A new electrocatalytic scheme for the detection of DNA is described. This scheme relies on the different interactions of single- and double-stranded oligonucleotides with the organic dyes methylene blue, thionin, azure A, azure B, toluidine blue O, brilliant cresyl blue and neutral red, which in addition can act as NADH catalysts. This catalytic activity can be used for amplifying differentiation between duplex and single-stranded DNA. Pyrolytic graphite electrodes modified with adsorbed dsDNA–organic dye complexes gave higher electrocatalytic NADH oxidation currents than those measured with electrodes modified with adsorbed ssDNA–organic dye. This difference is maximum for brilliant cresyl blue. These results suggest that the proposed scheme may be useful for the detection of DNA hybridisation and damage. Keywords: DNA hybridisation, Catalytic NADH oxidation, Phenoxazines, Phenothiazines, Phenazines

The electrochemical oxidation of 5′-polyadenylic acid (poly-A) on graphite electrodes has been studied by cyclic voltammetry. The oxidation of poly-A in neutral and alkaline solutions gives rise to redox-active products strongly adsorbed on the electrode surface, which exhibit catalytic activity toward NADH oxidation. The amount and properties of the catalyst are remarkably influenced by the pH of the oxidation medium. Depending on the conditions of catalyst formation, it is possible to reduce the overpotential for NADH oxidation by 300 mV. Amperometric detection of NADH using electrodes modified from different media were evaluated and compared. Electrodes modified from 0.1 M NaOH solutions containing the polynucleotide allow carrying out the amperometric detection of NADH at 50 mV (vs. Ag/AgCl), at pH 9, within a wide concentration range, 2.5×10−8−1×10−4 M, and with a detection limit of 1.1×10−8 M. These electrodes also exhibit very good stability and reproducibility.

Graphite electrodes modified with a drop-coated layer of polyethyleneimine (PEI) and adenosine diphosphate (ADP) displayed an electrocatalytic response to NADH after the adenine moiety of ADP was electrochemically oxidised. NADH can be detected amperometrically in alkaline solution (pH 9.0) at low applied potentials (+50 mV (Ag/AgCl)). Using a stationary electrode arrangement, linear response for NADH concentrations between 1.0×10 −8 and 1.0×10 −4 M was found, with a response time of 12 s and a detection limit of 8×10 −9 M. The electrode was applied to the amperometric monitoring of the reaction between lactate and NAD + catalysed by lactate dehydrogenase (LDH). A flow injection-amperometric method for the determination of LDH activity in human serum was developed. The method allows a fast and accurate discrimination between pathological and normal LDH activity levels, with a sampling rate of 40 h −1 . Quantitative results for a random set of human serum samples were found to be in good agreement with the standard spectrophotometric method.

Molecularly imprinted polyphosphazenes are presented as sensitive coatings for the detection of rifamycin SV in water. These membranes were combined with voltammetric transduction using glassy carbon (GC) working electrodes. The receptor layers were obtained by the drop-coating technique using tetrahydrofuran (THF) solutions containing 0.27 % (m/v) of the polymer and different amounts of template (0.03–0.17 %, m/v). Evaporation of THF results on a water insoluble membrane, which shows good adherence to the GC electrode surface. After removal of the template from the solid polymer membrane, the binding of rifamycin SV was examined by equilibrating the membrane in an aqueous solution of the template. The amount of bound rifamycin SV was measured by cyclic voltammetry or differential pulse voltammetry. Similar experiments were carried out with imprinted membranes using rifampicin as template. Less effective binding was obtained for rifamycin SV in the rifampicin-imprinted polymer. The rifamycin SV sensitive electrode, constructed using the corresponding molecularly imprinted polymer, allows rifamycin SV measurement in the concentration interval between 2.5×10−7 and 6.3×10−6 M.

An enzymatic biosensor for hydrogen peroxide based on a horseradish peroxidase (HRP)-ferrocene carbon paste modified electrode and coated with a layer of electrochemically generated poly(o-aminophenol) is reported. A linear calibration curve is obtained over the range 1×10 −8 M to 1×10 −5 M. The biosensor responds to hydrogen peroxide in a few seconds and has a detection limit of 8.5×10 −9 M. The response of the biosensor is diffusion controlled at low substrate concentrations. Flow injection assays of hydrogen peroxide at a sampling rate of 150 injections per hour with a relative standard deviation of 0.8% (50 samples) are possible. Applicability of the sensor for measurement of hydrogen peroxide in real samples (milk) was demonstrated.

A reagentless lactate amperometric biosensor, constructed by immobilising the enzymes glutamic pyruvic transaminase (GPT) and l-lactate dehydrogenase (LDH) together with its cofactor nicotinamide adenine dinucleotide (NAD+) in carbon paste using an electropolymerised poly(o-phenylenediamine) (PPD) film, is described. The response of the electrode is based on the electrocatalytic oxidation, at low applied potentials, of the enzymatically produced NADH by the PPD conducting film. This sensor construction was optimised, the final biosensor contained 80 units of LDH, 5 units of GPT and 0.1 mg of NAD+ mg−1 of carbon paste. Interference by direct electrochemical oxidation of easily oxidizable substances such as uric acid, l-ascorbic acid, l-cysteine, glutathione and paracetamol, are drastically reduced by covering the PPD-modified electrode with a second electrochemically synthesized, non-conducting poly(o-aminophenol) film. The bienzyme-double polymeric layermodified electrode at an applied potential of 0 V (vs. AgAgCl), gives a linear response for lactate in the range 6 × 10−7 M−8.5 × 10−5 M and a response time of 80 s. This sensor was used for the quantification of lactic acid in cider. Results compared favourably with a standard spectrophotometric method.

A glutamate biosensor was developed by electropolymerizing o -phenylenediamine on a dehydrogenase-NAD + modified carbon paste electrode. The amperometric response to glutamate was obtained by means of the electrocatalytic oxidation of the enzymatically produced NADH, at an applied potential close to 0 V (Ag/AgCl). After optimizing carbon paste composition, polymer thickness and operating variables, a linear amperometric response to glutamate was obtained within the concentration range 5·0×10 −6 –7·8×10 −5 M with a detection limit of 3·8×10 −6 M. The biosensor was applied to the determination of glutamate in chicken bouillon cubes. Good accuracy was found with respect to a reference enzymatic spectrophotometric method.

A reversed-phase liquid chromatographic method involving precolumn derivatization with 9-fluorenyl-methylchloroformate (FMOC) has been developed for the determination of dimethylamine (DMA) in groundwater. FMOC reacts rapidly with DMA under mild conditions, and the derivative is separated from matrix components and FMOC degradation products on a C18 column using isocratic elution with a mobile phase of 0.03 M acetate buffer (pH 4.0)-acetonitrile (30:70, v/v). Mean recovery was 98.5±4% and a detection limit of 4.5·10−7 M, corresponding to an injected amount of 3.6 pmol, was estimated using fluorimetric detection with excitation and emission wavelengths of 265 and 310 nm, respectively. No matrix interferences were found even when highly coloured and/or cloudy samples were examined. Several mixtures containing eleven amines, diamines and amino acids can be resolved using this method.

A detailed study of the electrochemistry of clenbuterol at bare carbon-paste electrodes (CPEs) has been carried out. Results showed that clenbuterol undergoes an ECE process. This compound is irreversibly oxidised at high potentials, resulting in the formation of a product which demonstrates quasi-reversible electrochemical behaviour at less positive potentials. The amount of this chemical product formed is very pH-dependent. Investigations into the electrochemical behaviour of clenbuterol at Nafion-modified CPEs were also made. The use of a thin Nafion film cast over the CPE resulted in a large increase in peak current over bare electrodes. Linear accumulation occurred with time, the linear range increasing with decreasing concentration. This allowed the detection of low concentrations of clenbuterol. Diffusion proved to be the rate-controlling process of clenbuterol through the Nafion membrane.

This chapter summarizes the major advances in the development of electrochemical sensors based on molecularly imprinted polymers as recognition element. A comprehensive and critical analysis of such chemical sensors, grouped around the different types of transduction is presented. The relevance of the type of transduction in defining the requirements for the design of MIPs and the different strategies for coupling MIPs to potentiometric, conductometric, impedimetric, voltammetric, and amperometric sensors are presented. The analytical characteristics of the different sensors are compiled in tables for easy comparison.

A horseradish peroxidase modified carbon paste electrode, useful for the determination of aniline at low ppb levels, is described. The enzyme was dispersed in the carbon paste and immobilised on a Nafion membrane. Cyclic voltammetry and differential pulse polarography were used to characterise the enzymatic electrochemical process, and several reaction products were identified. The electrode has been utilised for the flow injection amperometric detection of aniline at potentials down to +100 mV (Ag/AgCl), giving fast responses (t < 30 s), a measurement precision of 0.75% R.S.D. (n = 8) and a sampling frequency of 20 h−1. Application of the electrode to the analysis of aniline in spiked vegetable oil samples, using liquid-liquid extraction, resulted in a procedure giving no matrix interferences and a mean recovery of 99.4 ± 3.2%.

The preparation and application of novel mercury-coated caron fibre flow microelectrode are reported. The possibility to use this device in a flow system combined with alternating current (ac) stripping voltammetry is also demonstrated using mitoxantrone (MXT) as probe compound. The electrode is easy to prepare. Special analytical advantages have been observed when a.c. stripping voltammetry is applied to the adsorbed molecule on the electrode due to the reversibility of the electrochemical process. Once the operating system has been optimized a limit of detection for mitoxantrone of 4.5 × 10 −11 M was found. Manipulation of flow conditions allows the study of different concentration ranges. The direct analysis of serum samples without sample pretreatment, is also discussed and provided a linear calibration range between 4.5 × 10 −7 M and 4.5 × 10 −6 M.

The integrity of DNA purine bases was herein used to evaluate the antioxidant capacity. Unlike other DNA-based antioxidant sensors reported so far, the damaging agent chosen was the O 2 - radical enzymatically generated by the xanthine/xanthine oxidase system. An adenine-rich oligonucleotide was adsorbed on carbon paste electrodes and subjected to radical damage in the presence/absence of several antioxidant compounds. As a result, partial damage on DNA was observed. A minor product of the radical oxidation was identified by cyclic voltammetry as a diimine adenine derivative also formed during the electrochemical oxidation of adenine/guanine bases. The protective efficiency of several antioxidant compounds was evaluated after electrochemical oxidation of the remaining unoxidized adenine bases, by measuring the electrocatalytic current of NADH mediated by the adsorbed catalyst species generated. A comparison between O 2 - and OH radicals as a source of DNA lesions and the scavenging efficiency of various antioxidant compounds against both of them is discussed. Finally, the antioxidant capacity of beverages was evaluated and compared with the results obtained with an optical method.

Reactive oxygen species (ROS) are produced as a consequence of normal aerobic metabolism and are able to induce DNA oxidative damage. At the cellular level, the evaluation of the protective effect of antioxidants can be achieved by examining the integrity of the DNA nucleobases using electrochemical techniques. Herein, the use of an adenine-rich oligonucleotide (dA 21 ) adsorbed on carbon paste electrodes for the assessment of the antioxidant capacity is proposed. The method was based on the partial damage of a DNA layer adsorbed on the electrode surface by OH radicals generated by Fenton reaction and the subsequent electrochemical oxidation of the intact adenine bases to generate an oxidation product that was able to catalyze the oxidation of NADH. The presence of antioxidant compounds scavenged hydroxyl radicals leaving more adenines unoxidized, and thus, increasing the electrocatalytic current of NADH measured by differential pulse voltammetry (DPV). Using ascorbic acid (AA) as a model antioxidant species, the detection of as low as 50 nM of AA in aqueous solution was possible. The protection efficiency was evaluated for several antioxidant compounds. The biosensor was applied to the determination of the total antioxidant capacity (TAC) in beverages.

The conditions for the deposition of mercury on the surface of carbon fibers to permit the sensitive and reproducible voltammetry of Se(IV) have been investigated. Several procedures employing both predeposition and in situ formation of a mercury film on the carbon fiber were evaluated, and the effect of the mercury concentration and plating time upon the sensitivity of the stripping response for Se(IV) was determined. Due to the steady-state diffusional mass transport characteristics exhibited by these electrodes, the need for stirred solutions, a specific deposition potential, and a deposition time period in the stripping voltammetric procedure was eliminated. This procedure gave rise to a linear response up to 3 ng/ml, a detection limit of 0.11 ng/ml, and a relative standard deviation (RSD) of 4.4% at 5 ng/ml (n = 5) for determination of Se(IV).