Molecularly imprinted polymers - towards electrochemical sensors and electronic tongues.

Molecularly imprinted polymers (MIPs) are artificially synthesized materials to mimic the molecular recognition process of biological macromolecules such as substrate-enzyme or antigen-antibody. The combination of these biomimetic materials with electrochemical techniques has allowed the development of advanced sensing devices, which significantly improve the performance of bare or catalyst-modified sensors, being able to unleash new applications. However, despite the high selectivity that MIPs exhibit, those can still show some cross-response towards other compounds, especially with chemically analogous (bio)molecules. Thus, the combination of MIPs with chemometric methods opens the room for the development of what could be considered a new type of electronic tongues, i.e. sensor array systems,  based on its usage. In this direction, this review provides an overview of the more common synthetic approaches, as well as the strategies that can be used to achieve the integration of MIPs and electrochemical sensors, followed by some recent examples over different areas in order to illustrate the potential of such combination in very diverse applications.

Simultaneous Voltammetric Determination of Acetaminophen, Ascorbic Acid and Uric Acid by Use of Integrated Array of Screen-Printed Electrodes and Chemometric Tools.

In the present work, ternary mixtures of Acetaminophen, Ascorbic acid and Uric acid were resolved using the Electronic tongue (ET) principle and Cyclic voltammetry (CV) technique. The screen-printed integrated electrode array having differentiated response for the three oxidizable compounds was formed by Graphite, Prussian blue (PB), Cobalt (II) phthalocyanine (CoPc) and Copper oxide (II) (CuO) ink-modified carbon electrodes. A set of samples, ranging from 0 to 500 µmol·L-1, was prepared, using a tilted (33) factorial design in order to build the quantitative response model. Subsequently, the model performance was evaluated with an external subset of samples defined randomly along the experimental domain. Partial Least Squares Regression (PLS) was employed to construct the quantitative model. Finally, the model successfully predicted the concentration of the three compounds with a normalized root mean square error (NRMSE) of 1.00 and 0.99 for the training and test subsets, respectively, and R2 ≥ 0.762 for the obtained vs. expected comparison graphs. In this way, a screen-printed integrated electrode platform can be successfully used for voltammetric ET applications.

Voltammetric Electronic Tongue for the Simultaneous Determination of Three Benzodiazepines.

The presented manuscript reports the simultaneous detection of a ternary mixture of the benzodiazepines diazepam, lorazepam, and flunitrazepam using an array of voltammetric sensors and the electronic tongue principle. The electrodes used in the array were selected from a set of differently modified graphite epoxy composite electrodes; specifically, six electrodes were used incorporating metallic nanoparticles of Cu and Pt, oxide nanoparticles of CuO and WO3, plus pristine electrodes of epoxy-graphite and metallic Pt disk. Cyclic voltammetry was the technique used to obtain the voltammetric responses. Multivariate examination using Principal Component Analysis (PCA) justified the choice of sensors in order to get the proper discrimination of the benzodiazepines. Next, a quantitative model to predict the concentrations of mixtures of the three benzodiazepines was built employing the set of voltammograms, and was first processed with the Discrete Wavelet Transform, which fed an artificial neural network response model. The developed model successfully predicted the concentration of the three compounds with a normalized root mean square error (NRMSE) of 0.034 and 0.106 for the training and test subsets, respectively, and coefficient of correlation R ≥ 0.938 in the predicted vs. expected concentrations comparison graph.

Label-Free Aptasensor for Lysozyme Detection Using Electrochemical Impedance Spectroscopy.

This research develops a label-free aptamer biosensor (aptasensor) based on graphite-epoxy composite electrodes (GECs) for the detection of lysozyme protein using Electrochemical Impedance Spectroscopy (EIS) technique. The chosen immobilization technique was based on covalent bonding using carbodiimide chemistry; for this purpose, carboxylic moieties were first generated on the graphite by electrochemical grafting. The detection was performed using [Fe(CN)6]3-/[Fe(CN)6]4- as redox probe. After recording the frequency response, values were fitted to its electric model using the principle of equivalent circuits. The aptasensor showed a linear response up to 5 µM for lysozyme and a limit of detection of 1.67 µM. The sensitivity of the established method was 0.090 µM-1 in relative charge transfer resistance values. The interference response by main proteins, such as bovine serum albumin and cytochrome c, has been also characterized. To finally verify the performance of the developed aptasensor, it was applied to wine analysis.

A Voltammetric Electronic Tongue for the Resolution of Ternary Nitrophenol Mixtures.

This work reports the applicability of a voltammetric sensor array able to quantify the content of 2,4-dinitrophenol, 4-nitrophenol, and picric acid in artificial samples using the electronic tongue (ET) principles. The ET is based on cyclic voltammetry signals, obtained from an array of metal disk electrodes and a graphite epoxy composite electrode, compressed using discrete wavelet transform with chemometric tools such as artificial neural networks (ANNs). ANNs were employed to build the quantitative prediction model. In this manner, a set of standards based on a full factorial design, ranging from 0 to 300 mg·L-1, was prepared to build the model; afterward, the model was validated with a completely independent set of standards. The model successfully predicted the concentration of the three considered phenols with a normalized root mean square error of 0.030 and 0.076 for the training and test subsets, respectively, and r ≥ 0.948.

A novel electrochemical aptamer-antibody sandwich assay for lysozyme detection.

In this paper, we have reported a novel electrochemical aptamer-antibody based sandwich biosensor for the detection of lysozyme. In the sensing strategy, an anti-lysozyme aptamer was immobilized onto the carbon electrode surface by covalent binding via diazonium salt chemistry. After incubating with a target protein (lysozyme), a biotinylated antibody was used to complete the sandwich format. The subsequent additions of avidin-alkaline phosphatase as an enzyme label, and a 1-naphthyl phosphate substrate (1-NPP) allowed us to determine the concentration of lysozyme (Lys) via Differential Pulse Voltammetry (DPV) of the generated enzyme reaction product, 1-naphthol. Using this strategy, a wide detection range from 5 fM to 5 nM was obtained for a target lysozyme, with a detection limit of 4.3 fM. The control experiments were carried out by using bovine serum albumin (BSA), cytochrome c and casein. The results showed that the proposed biosensor had good specificity, stability and reproducibility for lysozyme analysis. In addition, the biosensor was applied for detecting lysozyme in spiked wine samples, and very good recovery rates were obtained in the range from 95.2 to 102.0% for lysozyme detection. This implies that the proposed sandwich biosensor is a promising analytical tool for the analysis of lysozyme in real samples.

Pd nanoparticles/multiwalled carbon nanotubes electrode system for voltammetric sensing of tyrosine.

A facile and quick synthesis of palladium decorated multi-walled carbon nanotubes is presented in this work. The developed protocol allowed a quasi-homogeneous distribution of the metal nanoparticles on the surface of the nanotubes, and a controlled size of the nanoparticles in a range between 3.5 and 4.5 nm. After the characterization of the hybrid nanocomposite a first attempt on a possible application was made. The preliminary test, an ink-like nanocomposite as a modifier on the surface of a carbon screen-printed electrode, was performed in order to detect L-Tyrosine. Preliminary results are promising. A catalytic effect on the oxidation peak of the L-Tyrosine was shown and furthermore a low limit of detection, 1.46 x 10(-10) M, was reached.

Avidin epoxy-graphite composite electrodes as platforms for genosensing and aptasensing.

This work presents two examples of biosensors that employ DNA as the recognition element. The transduction technique chosen is electrochemical impedance spectroscopy, which makes labeless detection possible. In the first case, a DNA probe was used to hybridize and detect a complementary DNA target; this principle may be used to construct biosensors to confirm a microbial, vegetal or animal species. The working example shown is the detection of enterohaemorragic Escherichia coli O104:H4 bacteria, blamed of an epidemic outbreak in vegetables in Germany in 2011. As a second example, a specific DNA aptamer able to interact with proteins, is used to develop a biosensor to detect thrombin, an important protein mediating in blood coagulation. Following the current trend in the field, these DNA biosensors have been prepared with help of nanocomponents in order to improve or to confirm and visualize the detection capabilities.

Label-free selective impedimetric detection of Cu2+ ions using catalytic DNA.

Copper is an essential element for regulation of many biological processes, however, in excess it is considered to be toxic for human health. This metal is frequently accompanied by other elements such as cadmium, nickel and lead. Thus, developing a selective and simple method for determination of copper in a matrix containing other heavy metal ions is of great importance. In this work, a novel selective method for copper detection was developed using electrodes modified with the DNAzyme capturing Cu(2+) ions. The DNAzyme reconstituted with copper catalyzes oxidation of ascorbic acid leading to the build-up and adsorption of oxidation products on the electrode surface and produces changes in the interfacial properties of the electrode. The increase in the interfacial electron-transfer resistance is probed with electrochemical impedance spectroscopy (EIS) in the presence of the reversible redox couple [Fe(CN)6](3-)/[Fe(CN)6](4-) as a marker. The DNAzyme based biosensor combines excellent selectivity against other heavy metal ions with sufficient sensitivity to Cu(2+) in the range of 6.5-40 µM.

A solid-contact ion selective electrode for copper(II) using a succinimide derivative as ionophore.

All-solid-state sensors with polyvinyl chloride (PVC)-based membranes using off-the-shelf N-hydroxysuccinimide (NHS) and succinimide (Succ) ionophores were prepared using DOP (dioctyl phthalate) and NPOE (ortho-nitrophenyloctyl ether) as plasticizers. Good responses were obtained when NHS was used. The potentiometric response of the proposed electrode is independent of pH over the range 2-6. The electrode shows a fast response time of 0.25 s. The electrode exhibits a Super-Nernstian response, with 37.5 mV/decade, with a potentiometric detection limit of 4.4 µM. The proposed sensor revealed good selectivity towards a group of transition metal ions.

A priori tailored selection of sensor arrays for electronic tongues.

Electronic tongues (ETs) are multisensor systems based on the coupling of different sensors with high stability and cross-sensitivity towards different species, along with an appropriate chemometric tool that allow the classification of samples and/or the quantification of selected analytes. Despite the great advantages that the data processing stage may offer (e.g. allowing to offset matrix effects from the sample itself, or to resolve the presence of interferences, drifts or non-linearities obtained with the sensors), the stability of the sensors response together with its varied cross-sensitivity are of utmost importance. In this regard, despite the progress that has been made over the last years and the increasing number of publications dealing with ETs, researchers have focused mainly on the developed application itself or on the data treatment stage, but very few report on the choice of the sensors. In this direction, herein we explore on the development of a simple methodology that allows the a priori selection of the optimal sensor array to carry out a specific application. Concretely, the usage of principal component analysis (PCA) in combination with different clustering indices is proposed as a simple approach to simultaneously assess the cross-response and the reproducibility of the different sensors in a single step, what allows to define which sensors may constitute an ET array for a given qualitative or quantitative application. The above ideas have been applied to the study case of aromatic essential oils.

Rapid field identification of subjects involved in firearm-related crimes based on electroanalysis coupled with advanced chemometric data treatment.

We demonstrate a novel system for the detection and discrimination of varying levels of exposure to gunshot residue from subjects in various control scenarios. Our aim is to address the key challenge of minimizing the false positive identification of individuals suspected of discharging a firearm. The chemometric treatment of voltammetric data from different controls using Canonical Variate Analysis (CVA) provides several distinct clusters for each scenario examined. Multiple samples were taken from subjects in controlled tests such as secondary contact with gunshot residue (GSR), loading a firearm, and postdischarge of a firearm. These controls were examined at both bare carbon and gold-modified screen-printed electrodes using different sampling methods: the 'swipe' method with integrated sampling and electroanalysis and a more traditional acid-assisted q-tip swabbing method. The electroanalytical fingerprint of each sample was examined using square-wave voltammetry; the resulting data were preprocessed with Fast Fourier Transform (FFT), followed by CVA treatment. High levels of discrimination were thus achieved in each case over 3 classes of samples (reflecting different levels of involvement), achieving maximum accuracy, sensitivity, and specificity values of 100% employing the leave-one-out validation method. Further validation with the 'jack-knife' technique was performed, and the resulting values were in good agreement with the former method. Additionally, samples from subjects in daily contact with relevant metallic constituents were analyzed to assess possible false positives. This system may serve as a potential method for a portable, field-deployable system aimed at rapidly identifying a subject who has loaded or discharged a firearm to verify involvement in a crime, hence providing law enforcement personnel with an invaluable forensic tool in the field.

Resolution of phenolic antioxidant mixtures employing a voltammetric bio-electronic tongue.

This work reports the application of a Bio-Electronic Tongue (BioET) system made from an array of enzymatic biosensors in the analysis of polyphenols, focusing on major polyphenols found in wine. For this, the biosensor array was formed by a set of epoxy-graphite biosensors, bulk-modified with different redox enzymes (tyrosinase and laccase) and copper nanoparticles, aimed at the simultaneous determination of the different polyphenols. Departure information was the set of voltammograms generated with the biosensor array, selecting some characteristic features in order to reduce the data for the Artificial Neural Network (ANN). Finally, after the ANN model optimization, it was used for the resolution and quantification of each compound. Catechol, caffeic acid and catechin formed the three-analyte case study resolved in this work. Good prediction ability was attained, therefore allowing the separate quantification of the three phenols with predicted vs. expected slope better than 0.970 for the external test set (n = 10). Finally, BioET has been also tested with spiked wine samples with good recovery yields (values of 104%, 117% and 122% for catechol, caffeic acid and catechin, respectively).

A reusable impedimetric aptasensor for detection of thrombin employing a graphite-epoxy composite electrode.

Here, we report the application of a label-free electrochemical aptasensor based on a graphite-epoxy composite electrode for the detection of thrombin; in this work, aptamers were immobilized onto the electrodes surface using wet physical adsorption. The detection principle is based on the changes of the interfacial properties of the electrode; these were probed in the presence of the reversible redox couple [Fe(CN)(6)](3-)/[Fe(CN)(6)](4-) using impedance measurements. The electrode surface was partially blocked due to formation of aptamer-thrombin complex, resulting in an increase of the interfacial electron-transfer resistance detected by Electrochemical Impedance Spectroscopy (EIS). The aptasensor showed a linear response for thrombin in the range of 7.5 pM to 75 pM and a detection limit of 4.5 pM. The aptasensor was regenerated by breaking the complex formed between the aptamer and thrombin using 2.0 M NaCl solution at 42 °C, showing its operation for different cycles. The interference response caused by main proteins in serum has been characterized.

A Sensor Array Based on Molecularly Imprinted Polymers and Machine Learning for the Analysis of Fluoroquinolone Antibiotics.

Fluoroquinolones (FQs) are one of the most important types of antibiotics in the clinical, poultry, and aquaculture industries, and their monitoring is required as the abuse has led to severe issues, such as antibiotic residues and antimicrobial resistance. In this study, we report a voltammetric electronic tongue (ET) for the simultaneous determination of ciprofloxacin, levofloxacin, and moxifloxacin in both pharmaceutical and biological samples. The ET comprises four sensors modified with three different customized molecularly imprinted polymers (MIPs) and a nonimprinted polymer integrated with Au nanoparticle-decorated multiwall carbon nanotubes (Au-fMWCNTs). MWCNTs were first functionalized to serve as a supporting substrate, while the anchored Au nanoparticles acted as a catalyst. Subsequently, MIP films were obtained by electropolymerization of pyrrole in the presence of the different target FQs. The sensors' morphology was characterized by scanning electron microscopy and transmission electron microscopy, while the modification process was followed electrochemically step by step employing [Fe(CN)6]3-/4- as the redox probe. Under the optimal conditions, the MIP(FQs)@Au-fMWCNT sensors exhibited different responses, limits of detection of ca. 1 µM, and a wide detection range up to 300 µM for the three FQs. Lastly, the developed ET presents satisfactory agreement between the expected and obtained values when used for the simultaneous determination of mixtures of the three FQs (R2 ≥0.960, testing subset), which was also applied to the analysis of FQs in commercial pharmaceuticals and spiked human urine samples.

A novel electronic tongue using electropolymerized molecularly imprinted polymers for the simultaneous determination of active pharmaceutical ingredients.

The combination of chemometrics and electrochemical sensors modified with molecularly imprinted polymers (MIPs) towards the development of MIP-based electronic tongues (ETs) was explored herein. To demonstrate the potential of such an approach, the simultaneous determination of paracetamol, ascorbic acid and uric acid mixtures in pharmaceutical samples was evaluated. To this aim, MIP-based sensors for the different compounds were prepared by in situ electropolymerization of pyrrole in the presence of p-toluenesulfonate anion (pTS-), which acted as functional doping ion of the polypyrrole (PPy) MIP backbone. Morphological characterization of the MIPs was done by scanning electron microscopy (SEM), while functionalization of the electrodes was monitored electrochemically. Under the optimized measuring conditions, the developed sensors showed a good performance, with good linearity at the µM level (R2 > 0.992, limits of detection between 1 and 24 µM) as well as good repeatability (intra- and inter-day RSD values between 3 and 6% over 30 consecutive measurements). Finally, the quantification of the individual substances in different pharmaceutical samples was achieved by an artificial neural networks (ANNs) model, showing satisfactory agreement between expected and obtained values (R2 > 0.987).

Bioinspired sensor systems.

This editorial summarizes and classifies the contributions presented by different authors to the special issue of the journal Sensors dedicated to Bioinspired Sensor Systems. From the coupling of sensor arrays or networks, plus computer processing abilities, new applications to mimic or to complement human senses are arising in the context of ambient intelligence. Principles used, and illustrative study cases have been presented permitting readers to grasp the current status of the field.

Potentiometric electronic tongue to resolve mixtures of sulfide and perchlorate anions.

This work describes the use of an array of potentiometric sensors and an artificial neural network response model to determine perchlorate and sulfide ions in polluted waters, by what is known as an electronic tongue. Sensors used have been all-solid-state PVC membrane selective electrodes, where their ionophores were different metal-phtalocyanine complexes with specific and anion generic responses. The study case illustrates the potential use of electronic tongues in the quantification of mixtures when interfering effects need to be counterbalanced: relative errors in determination of individual ions can be decreased typically from 25% to less than 5%, if compared to the use of a single proposed ion-selective electrode.

Graphene for the Building of Electroanalytical Enzyme-Based Biosensors. Application to the Inhibitory Detection of Emerging Pollutants.

Graphene and its derivates offer a wide range of possibilities in the electroanalysis field, mainly owing to their biocompatibility, low-cost, and easy tuning. This work reports the development of an enzymatic biosensor using reduced graphene oxide (RGO) as a key nanomaterial for the detection of contaminants of emerging concern (CECs). RGO was obtained from the electrochemical reduction of graphene oxide (GO), an intermediate previously synthesized in the laboratory by a wet chemistry top-down approach. The extensive characterization of this material was carried out to evaluate its proper inclusion in the biosensor arrangement. The results demonstrated the presence of GO or RGO and their correct integration on the sensor surface. The detection of CECs was carried out by modifying the graphene platform with a laccase enzyme, turning the sensor into a more selective and sensitive device. Laccase was linked covalently to RGO using the remaining carboxylic groups of the reduction step and the carbodiimide reaction. After the calibration and characterization of the biosensor versus catechol, a standard laccase substrate, EDTA and benzoic acid were detected satisfactorily as inhibiting agents of the enzyme catalysis obtaining inhibition constants for EDTA and benzoic acid of 25 and 17 mmol·L-1, respectively, and a maximum inhibition percentage of the 25% for the EDTA and 60% for the benzoic acid.

Integrating molecularly imprinted polymer beads in graphite-epoxy electrodes for the voltammetric biosensing of histamine in wines.

This manuscript presents a voltammetric biosensing study with use of molecularly imprinted polymers to detect histamine in wine. Polymer beads were synthesized by standard precipitation polymerization method and implemented on the electrode surface via sol-gel immobilization. Scanning and confocal microscopy examinations permitted characterizing the material. Adsorptive stripping voltammetry in differential mode was the technique chosen for final application, selecting an enrichment time of 5 min. These conditions permitted a limit of detection of 0.19 µg mL-1 (1.0 µM), with a linear response range from 0.5 to 6.0 µg mL-1 (2.71-32.4 µM). The repeatability of the measurements was 4.6% relative standard deviation (n = 12). Principal component analysis showed the ability of the prepared receptor for discriminating other biogenic amines and potential interfering species. A final application, illustrating the determination of histamine, was completed to show agreement of results between the fluorimetric reference method and the proposed electrochemical approach.