Multivariate Optimization of Electrochemical Biosensors for the Determination of Compounds Related to Food Safety-A Review.

We summarize the application of multivariate optimization for the construction of electrochemical biosensors. The introduction provides an overview of electrochemical biosensing, which is classified into catalytic-based and affinity-based biosensors, and discusses the most recent published works in each category. We then explore the relevance of electrochemical biosensors for food safety analysis, taking into account analytes of different natures. Then, we describe the chemometrics tools used in the construction of electrochemical sensors/biosensors and provide examples from the literature. Finally, we carefully discuss the construction of electrochemical biosensors based on design of experiments, including the advantages, disadvantages, and future perspectives of using multivariate optimization in this field. The discussion section offers a comprehensive analysis of these topics.

Development of a voltammetric electronic tongue for the simultaneous determination of synthetic antioxidants in edible olive oils.

A voltammetric electronic tongue (E-tongue) is "a multisensor system, which consists of a number of low-selective sensors and uses advanced mathematical procedures for signal processing based on pattern recognition and/or data multivariate analysis such as artificial neural networks (ANNs), principal component analysis (PCA), among others". Thus, E-tongues in combination with chemometrics tools result in more accurate and selective analytical methods. In this work, we report results of a simple and reliable electroanalytical method to determine butyl hydroxyanisole (BHA), butyl hydroxytoluene (BHT) and propyl gallate (PG) in edible olive oils (EOO). Therefore, the square wave voltammetry (SWV) was used on platinum and carbon fiber disk ultramicroelectrodes (E-tongue configuration) combined with chemometrics tools to perform these studies. On the other hand, two data fusion strategies were used in order to combine electrochemical data obtained for each working electrode in the E-tongue: low-level data fusion (LLDF) and mid-level data fusion (MLDF). In addition, to reduce the dimensionality of the dataset in MLDF, the discrete wavelet transform (DWT) was used. Finally, to assert the predictive capability of the method for BHA, BHT, and PG determination in real samples, a recovery study for the antioxidants in EOO samples was performed, demonstrating the analytical accuracy of the proposed method. Moreover, from the comparison between the proposed electrochemical method with the AOAC reference method and others found in the literature in terms of the quality of the model (REP %) and the percent recovery assays (%) in different samples, our results were better than other reported previously for the simultaneous determination of BHA, BHT, and PG in real samples. Moreover, the percent recovery assays obtained with the proposed electrochemical method were in good agreement with those obtained by the chromatographic method.

Development of an enzymatic biosensor to determine eugenol in dental samples.

A GCE/CRGO-ßCD's/ADA-SPE/AuNPs biosensor was successfully developed to determine eugenol in dental samples. The optimal conditions to construct the biosensor were obtained from an experimental design based on the response surfaces methodology. The GCE/CRGO-ßCD/ADA-SPE/AuNPs biosensor exhibited a very good analytical performance for the quantification of eugenol. Thus, it shows a linear range between 1.3 × 10-8 and 1 × 10-5 mol L-1, with a sensitivity of (5.3 ± 0.3) x 10-3 A mol-1 L. The limits of detection and quantification were 4 × 10-9 mol L-1 and 1.3 × 10-8 mol L-1, respectively. Biosensors had an intraday and inter day reproducibility of 5% and 8%, respectively. The repeatability was of 3%, and the stability was 21 days (a decrease of 30% in current responses was observed after the fourth week). Recovery studies were performed in order to validate the proposed method. Recovery percentages were between 94 and 108%. A value of the apparent Michaellis-Menten constant, KMapp, of 3.1 × 10-6 mol L-1 was obtained using both Lineweaver-Burk and Eadi-Hofstee methods.

Determination of kinetic parameters of the enzymatic reaction between soybean peroxidase and natural antioxidants using chemometric tools.

The oxidation of eugenol, isoeugenol and vanillin natural antioxidants catalyzed by the soybean peroxidase enzyme was studied using uv-vis spectroscopy. An experimental design was used to optimize the different variables. The multivariate curve resolution method was used to obtain the profiles of antioxidant absorbance's as a function of time due to uv-vis absorption bands of both antioxidants and the enzymatic reaction product/s show a strong overlap. From these results, apparent Michaelis-Menten constants as well as the kinetic parameters k1 and k3 involved in the catalytic cycle of peroxidases were calculated. The antioxidant apparent acidity constants were also determined at different pH's from uv-vis spectrophotometric measurements. Values of k1 were (0.6 ±â€¯0.1) × 105 M-1 s-1, (2.0 ±â€¯0.2) × 105 M-1 s-1 and (7.0 ±â€¯0.5) × 106 M-1 s-1 and k3 (4.0 ±â€¯0.2) × 105 M-1 s-1, (6.0 ±â€¯0.6) × 105 M-1 s-1 and (6.0 ±â€¯0.9) × 106 M-1 s-1 for eugenol, isoeugenol and vanillin, respectively.

Development of an electrochemical method to determine phenolic monoterpenes in essential oils.

A simple, rapid and non-expensive method is proposed to determine phenolic monoterpenes such as thymol and carvacrol in essential oils of thyme and oregano. The linear sweep voltammetry based on glassy carbon electrodes was the electrochemical technique used. Thymol and carvacrol have one main oxidation peak in non-aqueous media centered at about 1.3 V vs. Ag/AgCl. The electron transfer process is mainly diffusion controlled. The calibration plots generated using the commercial standards of thymol and carvacrol were used to estimate the total content in real samples. The calibration plots were linear in the concentration range from 8.5 × 10-5 to 1.3 × 10-3 mol L-1 and 7.9 × 10-5 to 1.2 × 10-3 mol L-1 for thymol and carvacrol, respectively. Results obtained with the electrochemical method are in good agreement with those of the official method (gas chromatography). In addition, the analytical procedure does not require previous preparation of the sample or modification of the electrode surface. The electrochemical technique used is very simple to apply. Under these conditions, the methodology proposed is a good, simple and fast option to perform a quality control of essential oils.

Sterigmatocystin: A mycotoxin to be seriously considered.

Sterigmatocystin is a carcinogenic compound that affects several species of crops and several species of experimental animals. The sterigmatocystin biosynthetic pathway is the best known and most studied. The International Agency for Research on Cancer classifies sterigmatocystin in the Group 2B. Three groups of analytical methods to determine sterigmatocystin in food can be found: chromatographic, ELISA immunoassays and chemical sensors. In addition, sterigmatocystin is a precursor of aflatoxin B1 in those cases where cereals and/or food are contaminated with fungi capable of producing aflatoxins. Chemical structures of sterigmatocystin and aflatoxin B1 are similar. These mycotoxins are pathogens of animals and cereals, producing a major economic impact on biotechnology and agricultural and food industries. This review summarizes different aspects related to sterigmatocystin such as its biosynthesis, toxicological studies and analytical methods for its determination.

Handling time misalignment and rank deficiency in liquid chromatography by multivariate curve resolution: Quantitation of five biogenic amines in fish.

Biogenic amines (BAs) are used for identifying spoilage in food. The most common are tryptamine (TRY), 2-phenylethylamine (PHE), putrescine (PUT), cadaverine (CAD) and histamine (HIS). Due to lack of chromophores, chemical derivatization with dansyl was employed to analyze these BAs using high performance liquid chromatography with a diode array detector (HPLC-DAD). However, the derivatization reaction occurs with any primary or secondary amine, leading to co-elution of analytes and interferents with identical spectral profiles, and thus causing rank deficiency. When the spectral profile is the same and peak misalignment is present on the chromatographic runs, it is not possible to handle the data only with Multivariate Curve Resolution and Alternative Least Square (MCR-ALS), by augmenting the time, or the spectral mode. A way to circumvent this drawback is to receive information from another detector that leads to a selective profile for the analyte. To overcome both problems, (tri-linearity break in time, and spectral mode), this paper proposes a new analytical methodology for fast quantitation of these BAs in fish with HPLC-DAD by using the icoshift algorithm for temporal misalignment correction before MCR-ALS spectral mode augmented treatment. Limits of detection, relative errors of prediction (REP) and average recoveries, ranging from 0.14 to 0.50 µg mL(-1), 3.5-8.8% and 88.08%-99.68%, respectively. These are outstanding results obtained, reaching quantification limits for the five BAs much lower than those established by the Food and Agriculture Organization of the United Nations and World Health Organization (FAO/WHO), and the European Food Safety Authority (EFSA), all without any pre-concentration steps. The concentrations of BAs in fish samples ranged from 7.82 to 29.41 µg g(-1), 8.68-25.95 µg g(-1), 4.76-28.54 µg g(-1), 5.18-39.95 µg g(-1) and 1.45-52.62 µg g(-1) for TRY, PHE, PUT, CAD, and HIS, respectively. In addition, the proposed method spends less than 4 min in an isocratic run, consuming less solvent in accordance with the principles of green analytical chemistry.

Development of a highly sensitive noncompetitive electrochemical immunosensor for the detection of atrazine by phage anti-immunocomplex assay.

The development of immunosensors for the detection of small molecules is of great interest because of their simplicity, high sensitivity and extended analytical range. Due to their size, small compounds cannot be simultaneously recognized by two antibodies impeding their detection by noncompetitive two-site immunoassays, which are superior to competitive ones in terms of sensitivity, kinetics, and working range. In this work, we combine the advantages of magneto-electrochemical immunosensors with the improved sensitivity and direct proportional signal of noncompetitive immunoassays to develop a new Phage Anti-Immunocomplex Electrochemical Immunosensor (PhAIEI) for the detection of the herbicide atrazine. The noncompetitive assay is based on the use of recombinant M13 phage particles bearing a peptide that specifically recognizes the immunocomplex of atrazine with an anti-atrazine monoclonal antibody. The PhAIEI performed with a limit of detection (LOD) of 0.2 pg mL(-1), which is 200-fold better than the LOD obtained using the same antibody in an optimized conventional competitive ELISA, with a large increase in working range. The developed PhAIEI was successfully used to assay undiluted river water samples with no pretreatment and excellent recoveries. Apart from the first demonstration of the benefits of integrating phage anti-immunocomplex particles into electrochemical immunosensors, the extremely low and environmentally relevant detection limits of atrazine attained with the PhAIEIS may have direct applicability to fast and sensitive detection of this herbicide in the environment.

Quantitative determination of tocopherols in edible vegetable oils using electrochemical ultra-microsensors combined with chemometric tools.

We have developed an electroanalytical method to quantify different isomers of tocopherols in edible vegetable oils. The method uses the square wave voltammetry on a carbon fiber disk ultramicroelectrode in benzene/ethanol+0.1 mol L(-1)H2SO4. Because the oxidation peaks of these natural antioxidants show an important overlapping, we have used two chemometric tools to obtain the multivariate calibration model. One method was the multivariate curve resolution-alternating least square (MCR-ALS), which assumes a linear behavior, i.e., the total signal is the sum of individual signals of components, and another nonlinear method such as artificial neuronal networks (ANNs). From the accuracy and precision analysis between nominal and estimated concentrations by both methods, we could infer that the ANNs method was a good model to quantify tocopherols in edible oil samples. Recovery percentages were between 94% and 99%. In addition, we found a difference of 1.4-6.8% between the total content of tocopherols in edible oil samples and the vitamin E content declared by the manufacturers.

Development of an amperometric biosensor based on peroxidases to quantify citrinin in rice samples.

An amperometric biosensor based on horseradish peroxidase (EC1.11.1.7,H2O2-oxide-reductases) to determine the content of citrinin mycotoxin in rice samples is proposed by the first time. The method uses carbon paste electrodes filled up with multi-walled carbon nanotubes embedded in a mineral oil, horseradish peroxidase, and ferrocene as a redox mediator. The biosensor is covered externally with a dialysis membrane, which is fixed to the body side of the electrode with a Teflon laboratory film, and an O-ring. The reproducibility and the repeatability were of 7.0% and 3.0%, respectively, showing a very good biosensor performance. The calibration curve was linear in a concentration range from 1 to 11.6nM. The limits of detection and quantification were 0.25nM and 0.75nM, respectively. For comparison, the citrinin content in rice samples was also determined by fluorimetric measurements. A very good correlation was obtained between the electrochemical and spectrophotometric methods.

Electrochemical oxidation of butein at glassy carbon electrodes.

The electrochemical oxidation of flavonoid butein is studied at glassy carbon electrodes in phosphate and citrate buffer solutions of different pH values, and 1M perchloric acid aqueous solutions by cyclic and square wave voltammetries. The oxidation peak corresponds to the 2e(-), 2H(+) oxidation of the 3,4-dihydroxy group in B ring of butein, given the corresponding quinone species. The overall electrode process shows a quasi-reversible behavior and an adsorption/diffusion mixed control at high butein bulk concentrations. At low butein concentrations, the electrode process shows mainly an adsorption control. Butein surface concentration values were obtained from the charge associated with the adsorbed butein oxidation peaks, which are in agreement with those values expected for the formation of a monolayer of adsorbate in the concentration range from 1 to 5µM. Square wave voltammetry was used to perform a full thermodynamic and kinetics characterization of the butein surface redox couple. Therefore, from the combination of the "quasi-reversible maximum" and the "splitting of the net square wave voltammetric peak" methods, values of (0.386±0.003) V, (0.46±0.04), and 2.7×10(2)s(-1) were calculated for the formal potential, the anodic transfer coefficient, and the formal rate constant, respectively, of the butein overall surface redox process in pH4.00 citrate buffer solutions. These results will be then used to study the interaction of butein, and other flavonoids with the deoxyribonucleic acid, in order to better understand the potential therapeutic applications of these compounds.

Ultra-sensitive electrochemical immunosensor using analyte peptidomimetics selected from phage display peptide libraries.

Immunosensors for small analytes have been a great addition to the analytical toolbox due to their high sensitivity and extended analytical range. In these systems the analyte is detected when it competes for binding to the detecting antibody with a tracer compound. In this work we introduce the use of phage particles bearing peptides that mimic the target analyte as surrogates for conventional tracers. As a proof of concept, we developed a magneto-electrochemical immunosensor (EI) for the herbicide molinate and compare its performance with conventional formats. Using the same anti-molinate antibody and phage particles bearing a molinate peptidomimetic, the EI performed with an IC(50) of 0.15 ngmL(-1) (linear range from 4.4 × 10(-3) to 10 ngmL(-1)). Compared to the conventional ELISA, the EI was faster (minutes), performed with a much wider linear range, and the detection limit that was 2500-fold lower. The EI produced consistent measurements and could be successfully used to assay river water samples with excellent recoveries. By using the same EI with a conventional tracer, we found that an important contribution to the gain in sensitivity is due to the filamentous structure of the phage (9 × 1000 nm) which works as a multienzymatic tracer, amplifying the competitive reaction. Since phage-borne peptidomimetics can be selected from phage display libraries in a straightforward systematic manner and their production is simple and inexpensive, they can contribute to facilitate the development of ultrasensitive biosensors.

Citrinin (CIT) determination in rice samples using a micro fluidic electrochemical immunosensor.

The development of an electrochemical immunosensor incorporated in a micro fluidic cell for quantification of citrinin (CIT) mycotoxin in rice samples is described for the first time. Both CIT present in rice samples and immobilized on a gold surface electrodeposited on a glassy carbon (GC) electrode modified with a cysteamine self-assembled monolayer were allowed to compete for the monoclonal mouse anti-CIT IgG antibody (mAb-CIT) present in solution. Then, an excess of rabbit anti mouse IgG (H+L) labelled with the horseradish peroxidase (secAb-HRP) was added, which reacts with the mAb-CIT which is in the immuno-complex formed with the immobilized CIT on the electrode surface. The HPR, in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the oxidation of catechol (H(2)Q) whose back electrochemical reduction was detected on a GC electrode at -0.15 V vs Ag/AgCl by amperometric measurements. The current measured is proportional to the enzymatic activity and inversely proportional to the amount of CIT present in the rice samples. This immunosensor for CIT showed a range of work between 0.5 and 50 ng mL(-1). The detection (LOD) and the quantification (LOQ) limits were 0.1 and 0.5 ng mL(-1), respectively. The coefficients of variation intra- and inter-assays were less than 6%. The electrochemical detection could be done within 2 min and the assay total time was 45 min. The immunosensor was provided to undertake at least 80 determinations for different samples with a minimum previous pre-treatment. Our electrochemical immunosensor showed a higher sensitivity and reduced analysis time compared to other analytical methods such as chromatographic methods. This methodology is fast, selective and very sensitive. Thus, the immunosensor showed to be a very useful tool to determine CIT in samples of cereals, mainly rice samples.

An amperometric biosensor based on peroxidases from Brassica napus for the determination of the total polyphenolic content in wine and tea samples.

An amperometric biosensor based on peroxidases from Brassica napus hairy roots (PBHR) used to determine the total polyphenolic content in wine and tea samples is proposed by the first time. The method employs carbon paste (CP) electrodes filled up with PBHR, ferrocene (Fc), and multi-walled carbon nanotubes embedded in a mineral oil (MWCNT+MO) at a given composition (PBHR-Fc-MWCNT+MO). The biosensor was covered externally with a dialysis membrane, which was fixed at the electrode body side part with a Teflon laboratory film and an O-ring. Calibration curves obtained from steady-state currents as a function of the concentration of a polyphenolic standard reference compound such as t-resveratrol (t-Res) or caffeic acid (CA) were then used to estimate the total polyphenolic content in real samples. The reproducibility and the repeatability were of 7.0% and 4.1% for t-Res (8.4% and 5.2% for CA), respectively, showing a good biosensor performance. The calibration curves were linear in a concentration range from 0.05 to 52 mg L(-1) and 0.06 to 69 mg L(-1) for t-Res and CA, respectively. The lowest polyphenolic compound concentration values measured experimentally for a signal to noise ratio of 3:1 were 0.023 mg L(-1) and 0.020 mg L(-1) for t-Res and CA, respectively.

Determination of progesterone (P4) from bovine serum samples using a microfluidic immunosensor system.

Progesterone (P4) is a steroidal hormone with a vital role in the maintenance of human and animal health. This paper describes the development of an immunosensor coupled to glassy carbon (GC) electrode and integrated to a microfluidic system to quantify P4 from bovine serum samples in a fast and sensitive way. The serum samples spiked with a given P4 concentration and a given P4 concentration bound to horseradish peroxide (HPR) were simultaneously added and, therefore, they competed immunologically with sheep monoclonal anti-P4 antibodies that were immobilized at a rotating disk. HRP in the presence of hydrogen peroxide (H(2)O(2)) catalyzes the chatecol (H(2)Q) oxidation to benzoquinone (Q). Its reverse electrochemical reduction to H(2)Q can be detected at a GC electrode surface at -0.15 V by chronoamperometric measurements. These current responses are proportional to the enzyme activity and inversely proportional to the P4 amount present in bovine serum samples. This P4 immunosensor showed a linear working range from 0.5 to 12.5 ng mL(-1). The detection (DL) and quantification (QL) limits were 0.2 and 0.5 ng mL(-1), respectively. The electrochemical immunosensor had a higher sensitivity than the ELISA method using conventional spectrophotometric detections. However, both methods allowed us to obtain similar detection limits. The immunosensor allowed us to make up to 100 determinations on different samples without any previous pre-treatment. This behavior proved to be suitable to detect P4 in routine veterinary, clinical, biological, physiological, and analytical assays.

Experimental study of the interplay between long-range electron transfer and redox probe permeation at self-assembled monolayers: evidence for potential-induced ion gating.

Evidence for the competition between long-range electron transfer across self-assembled monolayers (SAMs) and incorporation of the redox probe into the film is reported for the electroreduction of Ru(NH(3)) at hydroxyl- and carboxylic-acid-terminated SAMs on a mercury electrode, by using electrochemical techniques that operate at distinct time scales. Two limiting voltammetric behaviors are observed, consistent with a diffusion control of the redox process at mercaptophenol-coated electrodes and a kinetically controlled electron transfer reaction in the presence of neutral HS-(CH(2))(10)-COOH and HS-(CH(2))(n)()-CH(2)OH (n = 3, 5, and 10) SAMs. The monolayer thickness dependence of the standard heterogeneous electron transfer rate constant shows that the electron transfer plane for the reduction of Ru(NH(3)) at hydroxyl-terminated SAMs is located outside the film | solution interface at short times. However, long time scale experiments provide evidence for the occurrence of potential-induced gating of the adsorbed structure in some of the monolayers studied, which takes the form of a chronoamperometric spike. Redox probe permeation is shown to be a kinetically slow process, whose activation strongly depends on redox probe concentration, applied potential, and chemical composition of the intervening medium. The obtained results reveal that self-assembled monolayers made of mercaptobutanol and mercaptophenol preserve their electronic barrier properties up to the reductive desorption potential of a fully grown SAM, whereas those of mercaptohexanol, mercaptoundecanol, and mercaptoundecanoic acid undergo an order/disorder transition below a critical potential, which facilitates the approach of the redox probe toward the electrode surface.