María Pedrero

Amperometric flow-injection determination of phenolic compounds at self-assembled monolayer-based tyrosinase biosensors

The performance of a tyrosinase (Tyr) biosensor, constructed by immobilisation of the enzyme by cross-linking atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a Au disk electrode, is reported for the amperometric detection under flow-injection (FI) conditions of several phenolic compounds (phenol, catechol, m-cresol, p-cresol, 4-chloro-3-methylphenol, 3-chlorophenol, 4-chlorophenol, 2,4-dimethylphenol, 3,4-dimethylphenol, and 2-aminophenol). Experimental variables such as the detection potential (−100 mV versus Ag|AgCl|KCl 3 M), flow rate (1.02 ml min−1), injection volume (350 μl), and pH of the carrier solution (0.05 M phosphate buffer of pH 7.0) were optimised. Under these conditions, the Tyr biosensor exhibited a good reproducibility of the FI measurements, with no need to apply a cleaning or pre-treatment procedure. The useful lifetime of one single biosensor was 5 days. The kinetic parameters of the Tyr reaction were calculated for the 10 phenolic compounds. The analytical performance of the Tyr–MPA–Au electrode under flow through conditions was compared with that of other Tyr electrochemical biosensors reported in the literature. The usefulness of the biosensor for the analysis of real samples was proved by performing the estimation of the content of phenolic compounds in waste waters from a refinery at three different stages in the waste purification process.

Electrochemical activation of screen-printed carbon strips

Screen-printed electrodes, fabricated by thick-film technology, represent an attractive avenue for routine electrochemical sensing. However, the composite character of strips fabricated from commercial carbon strips results in slow rates of heterogeneous electron transfer. The aim of this study was to establish a rapid electrochemical procedure for in situ activation of screen-printed electrodes. Short pre-anodization periods were shown to increase the electrochemical activity for a wide range of irreversible and quasi-reversible redox processes. Activation parameters influencing the enhanced reversibility at strips fabricated from two common carbon inks were explored. The effect of the ink-curing temperature on the redox activity was also examined. Cyclic and differential-pulse voltammetry, X-ray photoelectron spectroscopy and scanning electron microscopy were used for monitoring changes in the electrochemical reversibility, surface area and morphology and the introduction of oxygen surface functionalities. The sensing utility of activated carbon strip electrodes is demonstrated for several analytes, and future prospects are discussed. The simple, yet effective, electrochemical pretreatment is compatible with on-site application of disposable electrodes.

Preparation, characterization and application of alkanethiol self-assembled monolayers modified with tetrathiafulvalene and glucose oxidase at a gold disk electrode

Abstract A critical study of the different variables affecting the preparation of enzyme electrochemical biosensors using self-assembled monolayer (SAM)-modified electrodes is reported. Regarding variables affecting the obtention of SAMs on a gold disk electrode (AuE), the type of alkanethiol employed, its concentration, the time and temperature of formation and the composition of the medium were evaluated. Concerning a glucose biosensor based on SAM-modified AuEs, the performances of several redox mediators for the enzymatic oxidation of glucose were compared. Two glucose oxidase (GOx) immobilization methods on the SAM-modified AuE were tested: a covalent binding using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N -hydroxysulfosuccinimide (NHSS), and a cross-linking with glutaraldehyde. The cross-linking method produced slopes of the substrate calibration graphs at a mercaptopropionic acid (MPA)-modified electrode two orders of magnitude higher than those obtained with the covalent binding. Moreover, the mediator tetrathiafulvalene (TTF) was co-immobilized atop the SAM together with GOx. A heterogeneous electron transfer constant between TTF and the MPA–GOx bioelectrode of 1.25 s −1 was calculated. The enzyme loading, the amount of TTF on the electrode, the applied potential and the pH were also optimized. A good repeatability of the measurements with the TTF–GOx–MPA–AuE biosensor was demonstrated, with no need of pretreatment of the modified electrode. No significant changes in the slope value for the glucose calibration graph were found after 5 days when working with the same biosensor. An apparent Michaelis–Menten constant of (13.9±0.5) mM, and a limit of detection for glucose of 3.5×10 −6 M were obtained. Moreover, the TTF–GOx–MPA–AuE also performed well in the flow-injection mode and in the analysis of glucose in real samples.

Disposable magnetic DNA sensors for the determination at the attomolar level of a specific Enterobacteriaceae family gene

Disposable magnetic DNA sensors using an enzyme-amplified strategy for the specific detection of a gene related to the Enterobacteriaceae bacterial family, based on the coupling of streptavidin-peroxidase to biotinylated lacZ gene target sequences, has been developed. A biotinylated 25-mer capture probe was attached to streptavidin-modified magnetic beads and hybridization with the biotinylated target was allowed to proceed. Then, a streptavidin-peroxidase polymer was attached to the biotinylated target, and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene (TTF) modified gold screen-printed electrodes (Au/SPEs). The amperometric response obtained at -0.15 V after the addition of hydrogen peroxide was used to detect the hybridization process. In order to improve the sensitivity of the determination and reduce the assay time, different variables of the assay protocol were optimized. A low detection limit (5.7 fmol) with good stability (RSD = 7.1%, n = 10) was obtained. The DNA nonspecific adsorption at the magnetic beads was negligible, the obtained results thus demonstrating the possibility to detect the hybridization event with great specificity and sensitivity. The developed method was used for the analysis of Escherichia coli DNA fragments (326 bases) in polymerase chain reaction (PCR) amplicons extracted from a cell culture. As low as 2.5 aM asymmetric PCR product could be detected with the developed methodology.

A peroxidase-tetrathiafulvalene biosensor based on self-assembled monolayer modified Au electrodes for the flow-injection determination of hydrogen peroxide

The construction and performance under flow-injection conditions of an integrated amperometric biosensor for hydrogen peroxide is reported. The design of the bioelectrode is based on a mercaptopropionic acid (MPA) self-assembled monolayer (SAM) modified gold disk electrode on which horseradish peroxidase (HRP, 24.3U) was immobilized by cross-linking with glutaraldehyde together with the mediator tetrathiafulvalene (TTF, 1mumol), which was entrapped in the three-dimensional aggregate formed. The amperometric biosensor allows the obtention of reproducible flow injection amperometric responses at an applied potential of 0.00V in 0.05molL(-1) phosphate buffer, pH 7.0 (flow rate: 1.40mLmin(-1), injection volume: 150muL), with a range of linearity for hydrogen peroxide within the 2.0x10(-7)-1.0x10(-4)molL(-1) concentration range (slope: (2.33+/-0.02)x10(-2)Amol(-1)L, r=0.999). A detection limit of 6.9x10(-8)molL(-1) was obtained together with a R.S.D. (n=50) of 2.7% for a hydrogen peroxide concentration level of 5.0x10(-5)molL(-1). The immobilization method showed a good reproducibility with a R.S.D. of 5.3% for five different electrodes. Moreover, the useful lifetime of one single biosensor was estimated in 13 days. The SAM-based biosensor was applied for the determination of hydrogen peroxide in rainwater and in a hair dye. The results obtained were validated by comparison with those obtained with a spectrophotometric reference method. In addition, the recovery of hydrogen peroxide in sterilised milk was tested.

Electroanalytical Sensors and Devices for Multiplexed Detection of Foodborne Pathogen Microorganisms

The detection and identification of pathogen microorganisms still rely on conventional culturing techniques, which are not suitable for on-site monitoring. Therefore, a great research challenge in this field is focused on the need to develop rapid, reliable, specific, and sensitive methods to detect these bacteria at low cost. Moreover, the growing interest in biochip development for large scale screening analysis implies improved miniaturization, reduction of analysis time and cost, and multi-analyte detection, which has nowadays become a crucial challenge. This paper reviews multiplexed foodborne pathogen microorganisms detection methods based on electrochemical sensors incorporating microarrays and other platforms. These devices usually involve antibody-antigen and DNA hybridization specific interactions, although other approaches such as the monitoring of oxygen consumption are also considered.

Electrochemical genosensors for the detection of cancer-related miRNAs

MicroRNAs (miRNAs), new stars of human genetics, are naturally occurring, 19−25 base pair, noncoding RNAs that regulate gene expression posttranscriptionally and have been demonstrated to be excellent biomarkers for cancer diagnosis/prognosis. Because of their short length, sequence similarity, and very low concentration, their detection in real samples is challenging. Among other methods for miRNA detection, electrochemical nucleic acid biosensors exhibit relevant advantages in terms of high sensitivity, ease of use, short assay time, nontoxic experimental steps, and adaptability to point-of-care testing. This article gives a brief overview of recent advances in the rapidly developing area of electrochemical biosensors for miRNA detection. The fundamentals of the different strategies developed to achieve novel signal amplification and sensitive electrochemical detection are discussed, and some examples of relevant approaches are highlighted, along with future prospects and challenges.

Disposable amperometric magnetoimmunosensors for the specific detection of Streptococcus pneumoniae

Disposable amperometric magnetoimmunosensors, based on the use of functionalized magnetic beads and gold screen-printed electrodes, have been developed for the selective detection and quantification of Streptococcus pneumoniae. A specific antibody prepared against a serotype 37 S. pneumoniae strain, selected by flow cytometry among seven anticapsular or antisomatic antibodies, was linked to Protein A-modified magnetic beads and incubated with bacteria. The same antibody, conjugated with horseradish peroxidase, was attached to the bacteria and the resulting modified magnetic beads were captured by a magnetic field on the surface of tetrathiafulvalene-modified gold screen-printed electrodes. The amperometric response obtained at -0.15 V vs. the silver pseudoreference electrode of the Au/SPE after the addition of H(2)O(2) was used as transduction signal. Different assay formats were examined and the experimental variables optimized. The limits of detection achieved, without pre-concentration or pre-enrichment steps, were 1.5×10(4) cfu mL(-1) (colony forming unit) and 6.3×10(5) cfu mL(-1) for S. pneumoniae strains Dawn (serotype 37) and R6 (non-encapsulated), respectively. The developed methodology shows a good selectivity against closely related streptococci and its usefulness for the analysis of inoculated urine samples has been demonstrated. The total analysis time of 3.5 h from sampling to measurement, the possibility to prepare up to 30 sensors per day and the use of small amounts of test solution for S. pneumoniae identification, constitute important advantages that make the developed methodology a promising alternative for clinical diagnosis.

Immunosensor for the determination of Staphylococcus aureus using a tyrosinase–mercaptopropionic acid modified electrode as an amperometric transducer

AbstractAn amperometric immunosensor for the quantification of Staphylococcus aureus based on the coimmobilization of rabbit immunoglobulin G (RbIgG) and tyrosinase on a mercaptopropionic acid self-assembled monolayer modified gold electrode is reported. A competitive mode in which protein-A-bearing S. aureus cells and antiRbIgG labeled with alkaline phosphatase (AP) compete for the binding sites of immobilized RbIgG was used. Monitoring of the affinity reaction was carried out by the amperometric detection at –0.15 V of phenol generated in the enzyme reaction with AP, at the tyrosinase-modified electrode through the electrochemical reduction of the o-quinone formed. Optimization of the working variables, such as the immunosensor composition and incubation times, the applied potential, the working pH and the concentration of phenyl phosphate used as the AP substrate, was carried out. Under the optimized conditions, both the repeatability of the measurements and the reproducibility of the responses obtained with different immunosensors yielded relative standard deviation values for the steady-state current lower than 10%. The immunosensor showed a dynamic range from 4.4×105 to 1.8×107S. aureus cells mL−1, with a detection limit of 1.7×105 cells mL−1. The limit of detection was remarkably improved by subjecting S. aureus cells to wall lysis by heat treatment. The value obtained was 2.3×103 cells mL−1, which is adequate for the monitoring of S. aureus contamination levels in some foodstuffs. As an application, milk samples spiked with bacteria at the 4.8×103 cells mL−1 level were analyzed. FigureThe immunosensor configuration. AP alkaline phosphatase, RbIgG rabbit immunoglobulin G, MPA mercaptopropionic acid

Electrochemical genosensors based on PCR strategies for microorganisms detection and quantification

The use of DNA electrochemical sensors combined with PCR-amplification strategies for the detection of microorganisms is reviewed. Most studies involve testing sensors for short oligonucleotide targets without DNA preparation from real samples. Approaches involving amperometric, voltammetric or impedimetric detection of hybridization processes between surface-attached DNA probes and targets are addressed. Direct detection of the hybridization process and the use of electrochemical redox mediators, enzyme amplification or nanoparticle-labeling are considered.