María Jesús Lobo-Castañón

Electrochemical sensors based on molecularly imprinted polymers

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.

Voltammetric sensor for vanillylmandelic acid based on molecularly imprinted polymer-modified electrodes

Despite the increasing number of applications of molecularly imprinted polymers (MIPs) in analytical chemistry, the construction of a biomimetic voltammetric sensor remains still challenging. This work investigates the development of a voltammetric sensor for vanillylmandelic acid (VMA) based on acrylic MIP-modified electrodes. Thin layers of MIPs for VMA have been prepared by spin coating the surface of a glassy carbon electrode with the monomers mixture (template, methacrylic acid, a cross-linking agent and solvent), followed by in situ photopolymerisation. After extraction of the template molecule, the peak current recorded with the imprinted sensor after rebinding was linear with VMA concentration in the range 19-350 microg ml(-1), whereas the response of the control electrode is independent of incubation concentration, and was about one-tenth of the value recorded with the imprinted sensor at the maximum concentration tested. Under the conditions used, the sensor is able to differentiate between VMA and other closely structural-related compounds, such as 3-methoxy-4-hydroxyphenylethylene glycol (not detected), or 3,4- and 2,5-dihydroxyphenilacetic acids, which are adsorbed on the bare electrode surface but not at the polymer layer. Homovanillic acid was detected with the imprinted sensors after incubation, indicating that the presence of both methoxy and carboxylic groups in the same position as in VMA is necessary for effective binding in the imprinted sites. Nevertheless, both species can be differentiated by the oxidation potential. It can be concluded that MIP-based voltammetric electrodes are very promising analytical tool for the development of highly selective analytical sensors.

Aptamer-based analysis: a promising alternative for food safety control.

Ensuring food safety is nowadays a top priority of authorities and professional players in the food supply chain. One of the key challenges to determine the safety of food and guarantee a high level of consumer protection is the availability of fast, sensitive and reliable analytical methods to identify specific hazards associated to food before they become a health problem. The limitations of existing methods have encouraged the development of new technologies, among them biosensors. Success in biosensor design depends largely on the development of novel receptors with enhanced affinity to the target, while being stable and economical. Aptamers fulfill these characteristics, and thus have surfaced as promising alternatives to natural receptors. This Review describes analytical strategies developed so far using aptamers for the control of pathogens, allergens, adulterants, toxins and other forbidden contaminants to ensure food safety. The main progresses to date are presented, highlighting potential prospects for the future.

A bienzyme-poly-(o-phenylenediamine)-modified carbon paste electrode for the amperometric detection of l-lactate

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.

DNA-based biosensor for the electrocatalytic determination of antioxidant capacity in beverages.

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.

Amperometric glutamate biosensor based on poly(o-phenylenediamine) film electrogenerated onto modified carbon paste electrodes

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 x 10(-6)-7.8 x 10(-5) M with a detection limit of 3.8 x 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.

Impedimetric aptasensor for tobramycin detection in human serum.

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.

Strongly structured DNA sequences as targets for genosensing: Sensing phase design and coupling to PCR amplification for a highly specific 33-mer gliadin DNA fragment

Electrochemical genosensors are becoming cost-effective miniaturizable alternatives to real-time PCR (RT-PCR) methods for the detection of sequence-specific DNA fragments. We report on the rapid detection of PCR amplicons without the need of purification or strand separation. A challenging target sequence for both PCR amplification and electrochemical detection allowed us to address some difficulties associated to hybridization on electrode surfaces. The target was a highly specific oligonucleotide sequence of wheat encoding the most immunogenic peptide of gliadin that triggers the immune response of celiac disease (CD), the 33-mer. With a sandwich assay format and a rational design of the capture and tagged-signaling probes the problems posed by the strong secondary structure of the target and complementary probes were alleviated. Using a binary self-assembled monolayer and enzymatic amplification, a limit of detection of 0.3 nM was obtained. The genosensor did not respond to other gluten-containing cereals such as rye and barley. Coupling to PCR to analyze wheat flour samples required tailoring both the capture and signaling probes. This is the first time that deleterious steric hindrance from long single-stranded regions adjacent to the electrode surface is reported for relatively short amplicons (less than 200 bp). The importance of the location of the recognition site within the DNA sequence is discussed. Since the selected gene fragment contains several repetitions of short sequences, a careful optimization of the PCR conditions had to be performed to circumvent the amplification of non-specific fragments from wheat flour.

Molecularly Imprinted Polyphosphazene Films as Recognition Element in a Voltammetric Rifamycin SV Sensor

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.

Targeting Helicase-Dependent Amplification Products with an Electrochemical Genosensor for Reliable and Sensitive Screening of Genetically Modified Organisms

Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.