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.

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).

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.

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.

Modification of electrodes with N-and S-doped carbon dots. Evaluation of the electrochemical response.

Nitrogen and sulphur-doped Carbons Dots (N-CDs and S-CDs) were synthesized by a hydrothermal method and incorporated as surface electrode modifiers to evaluate their properties for electrochemical sensing. The first task was to characterize the synthesized materials, for which different spectroscopies, scanning microscopes, mass spectrometry and elementary analysis were performed. Next, a glassy carbon electrode (GCE) was surface-modified with the doped CDs and applied to check the electrochemical signal of different organic compounds corresponding to different families. Water solubility of the doped carbon dots forced us to incorporate them in a graphite-polystyrene ink to complete the modification of electrodes. This modification needed a first activation to obtain a properly conductive surface. The organic compounds examined were salicylic acid, cysteine and ascorbic acid. The modified GCEs exhibited an enhanced sensitivity, probably caused by the increase of active surface, but in addition, signals of salicylic acid were shifted ca. 200 mV to lower potentials, what is a proof of the increase of the heterogeneous electron transfer rate, and a demonstration of an enhanced catalytic response.

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.

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.

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.

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.

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.

Voltammetric electronic tongue to identify Brett character in wines. On-site quantification of its ethylphenol metabolites.

This work reports the applicability of a voltammetric sensor array able to evaluate the content of the metabolites of the Brett defect: 4-ethylphenol, 4-ethylguaiacol and 4-ethylcatechol in spiked wine samples using the electronic tongue (ET) principles. The ET used cyclic voltammetry signals, obtained from an array of six graphite epoxy modified composite electrodes; these were compressed using Discrete Wavelet transform while chemometric tools, among these artificial neural networks (ANNs), were employed to build the quantitative prediction model. In this manner, a set of standards based on a modified full factorial design and ranging from 0 to 25mgL-1 on each phenol, was prepared to build the model; afterwards, the model was validated with an external test set. The model successfully predicted the concentration of the three considered phenols with a normalized root mean square error of 0.02 and 0.05, for the training and test subsets respectively, and correlation coefficients better than 0.958.

Electronic tongue for nitro and peroxide explosive sensing.

This work reports the application of a voltammetric electronic tongue (ET) towards the simultaneous determination of both nitro-containing and peroxide-based explosive compounds, two families that represent the vast majority of compounds employed either in commercial mixtures or in improvised explosive devices. The multielectrode array was formed by graphite, gold and platinum electrodes, which exhibited marked mix-responses towards the compounds examined; namely, 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX), pentaerythritol tetranitrate (PETN), 2,4,6-trinitrotoluene (TNT), N-methyl-N,2,4,6-tetranitroaniline (Tetryl) and triacetone triperoxide (TATP). Departure information was the set of voltammograms, which were first analyzed by means of principal component analysis (PCA) allowing the discrimination of the different individual compounds, while artificial neural networks (ANNs) were used for the resolution and individual quantification of some of their mixtures (total normalized root mean square error for the external test set of 0.108 and correlation of the obtained vs. expected concentrations comparison graphs r>0.929).

Three different signal amplification strategies for the impedimetric sandwich detection of thrombin.

In this work, we report a comparative study on three highly specific amplification strategies for the ultrasensitive detection of thrombin with the use of aptamer sandwich protocol. The protocol consisted on the use of a first thrombin aptamer immobilized on the electrode surface, the recognition of thrombin protein, and the reaction with a second biotinylated thrombin aptamer forming the sandwich. Through the exposed biotin end, three variants have been tested to amplify the electrochemical impedance signal. The strategies included (a) silver enhancement treatment, (b) gold enhancement treatment and (c) insoluble product produced by the combination of the enzyme horseradish peroxidase (HRP) and 3-amino-9-ethylcarbazole (AEC). The properties of the sensing surface were probed by electrochemical impedance measurements in the presence of the ferrocyanide/ferricyanide redox marker. Insoluble product strategy and silver enhancement treatment resulted in the lowest detection limit (0.3 pM), while gold enhancement method resulted in the highest reproducibility, 8.8% RSD at the pM thrombin concentration levels. Results of silver and gold enhancement treatment also permitted direct inspection by scanning electron microscopy (SEM).

A novel bio-electronic tongue using different cellobiose dehydrogenases to resolve mixtures of various sugars and interfering analytes.

A novel application of cellobiose dehydrogenase (CDH) as sensing element for a Bioelectronic Tongue (BioET) system has been tested. In this work CDHs from various fungi, which exhibit different substrate specificities, were used to discriminate between lactose and glucose in presence of the interfering matrix compound Ca(2+) in various mixtures. This work exploits the advantage of an electronic tongue system with practically zero pre-treatment of samples and operation at low voltages in a direct electron transfer mode. The Artificial Neural Network (ANN) used in the BioET system to interpret the voltammetric data was able to provide a correct prediction of the concentrations of the analytes considered. Correlation coefficients in the comparison of obtained vs. expected concentrations were highly significant, especially for lactose (R(2)=0.975) and Ca(2+) (R(2)=0.945). This BioET application has a high potential especially for the food and dairy industry and also, if further miniaturised in screen printed format, for its in-situ use.

Independent comparison study of six different electronic tongues applied for pharmaceutical analysis.

Electronic tongue technology based on arrays of cross-sensitive chemical sensors and chemometric data processing has attracted a lot of researchers' attention through the last years. Several so far reported applications dealing with pharmaceutical related tasks employed different e-tongue systems to address different objectives. In this situation, it is hard to judge on the benefits and drawbacks of particular e-tongue implementations for R&D in pharmaceutics. The objective of this study was to compare the performance of six different e-tongues applied to the same set of pharmaceutical samples. For this purpose, two commercially available systems (from Insent and AlphaMOS) and four laboratory prototype systems (two potentiometric systems from Warsaw operating in flow and static modes, one potentiometric system from St. Petersburg, one voltammetric system from Barcelona) were employed. The sample set addressed in the study comprised nine different formulations based on caffeine citrate, lactose monohydrate, maltodextrine, saccharin sodium and citric acid in various combinations. To provide for the fair and unbiased comparison, samples were evaluated under blind conditions and data processing from all the systems was performed in a uniform way. Different mathematical methods were applied to judge on similarity of the e-tongues response from the samples. These were principal component analysis (PCA), RV' matrix correlation coefficients and Tuckers congruency coefficients.

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.

Crown ether-modified electrodes for the simultaneous stripping voltammetric determination of Cd(II), Pb(II) and Cu(II).

This work describes the immobilization of 4-carboxybenzo-18-crown-6 (CB-18-crown-6) and 4-carboxybenzo-15-crown-5 (CB-15-crown-5) assisted by lysine on aryl diazonium salt monolayers anchored to the surface of graphite-epoxy composite electrodes (GEC), and their use for the simultaneous determination of Cd(II), Pb(II) and Cu(II) by differential pulse anodic stripping voltammetry (DPASV). These modified electrodes display a good repeatability and reproducibility with detection and quantification limits at levels of µg L(-1) (ppb), confirming their suitability for the determination of Cd(II), Pb(II) and Cu(II) ions in environmental samples. The overlapped nature of the multimetal stripping measurements was resolved by employing the two-sensor array CB-15-crown-5-GEC and CB-18-crown-6-GEC, since the metal complex selectivity exhibited by the considered ligands could add some discrimination power. For the processing of the voltammograms, Discrete Wavelet Transform and Causal Index were selected as preprocessing tools for data compression coupled with an artificial neural network for the modeling of the obtained responses, allowing the resolution of mixtures of these metals with good prediction of their concentrations (correlation with expected values for an external test subset better than 0.942).

DNA polymorphism sensitive impedimetric detection on gold-nanoislands modified electrodes.

Nanocomposite materials are being increasingly used in biosensing applications as they can significantly improve biosensor performance. Here we report the use of a novel impedimetric genosensor based on gold nanoparticles graphite-epoxy nanocomposite (nanoAu-GEC) for the detection of triple base mutation deletion in a cystic-fibrosis (CF) related human DNA sequence. The developed platform consists of chemisorbing gold nano-islands surrounded by rigid, non-chemisorbing, and conducting graphite-epoxy composite. The ratio of the gold nanoparticles in the composite was carefully optimized by electrochemical and microscopy studies. Such platform allows the very fast and stable thiol immobilization of DNA probes on the gold islands, thus minimizing the steric and electrostatic repulsion among the DNA probes and improving the detection of DNA polymorphism down to 2.25fmol by using electrochemical impedance spectroscopy. These findings are very important in order to develop new and renewable platforms to be used in point-of-care devices for the detection of biomolecules.