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This paper describes the results obtained in the development of the first electrochemical immunosensor described to date for the detection of E‐cadherin (E‐cad) protein, a relevant biomarker of prognosis and metastasis in cancer, based on the use of magnetic microcarriers (MBs) and amperometric transduction at screen‐printed carbon electrodes (SPCEs). Thus, the determination of E‐cad protein involved the use of two specific antibodies against this protein (one of them labelled with HRP) in a sandwich configuration onto HOOC‐MBs. The magnetic bioconjugates were captured onto SPCEs and the amperometric transduction was performed using the H2O2/hydroquinone (HQ) system. Under optimal conditions, this bioplatform demonstrated a wide linear concentration range (0.50–25 ng mL−1) and a detection limit as low as 0.16 ng mL−1, well below the optimal cut‐off level for the E‐cad protein (defined as 10,000 ng mL−1 for soluble E‐cad levels in serum). The developed sensor also showed a good reproducibility among measurements with seven different sensors constructed in the same manner (RSD, 5.4 %), stability for more than 15 days and good specificity towards other proteins commonly found on biological samples. The applicability of this simple handling bioplatform for the direct determination of this protein in cell lysates with different metastatic potential and extracts from paraffined‐embedded human colorectal cancer tissues of different grade were also demonstrated., The financial support of the Spanish Ministerio de Economía y Competitividad (CTQ2015‐64402‐C2‐1‐R Project) and the NANOAVANSENS Program from the Comunidad de Madrid (S2013/MT‐3029 Project) are gratefully acknowledged. C.M.S.M. was supported by a contract of the S2013/MT‐3029 Project. R.B. acknowledges the financial support of the PI17CIII/00045 grant from the AES‐ISCIII program. M.G.A. was supported by a contract of the Programa Operativo de Empleo Juvenil y la Iniciativa de Empleo Juvenil (YEI) with the participation of the Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid y del Fondo Social Europeo.

New amperometric immune-scaffolds for the rapid, sensitive and reliable determination of [...]

An amperometric magnetoimmunosensor for the determination of human p53 protein is described in this work using a sandwich configuration involving the covalent immobilization of a specific capture antibody onto activated carboxylic-modified magnetic beads (HOOC-MBs) and incubation of the modified MBs with a mixture of the target protein and horseradish peroxidase-labeled antibody (HRP-anti-p53). The resulting modified MBs are captured by a magnet placed under the surface of a disposable carbon screen-printed electrode (SPCE) and the amperometric responses are measured at −0.20 V (vs. an Ag pseudo-reference electrode), upon addition of hydroquinone (HQ) as a redox mediator and H2O2 as the enzyme substrate. The magnetoimmunosensing platform was successfully applied for the detection of p53 protein in different cell lysates without any matrix effect after a simple sample dilution. The results correlated accurately with those provided by a commercial ELISA kit, thus confirming the immunosensor as an attractive alternative for rapid and simple determination of this protein using portable and affordable instrumentation.

A bienzyme biosensor for the simultaneous determination of glucose and fructose was developed by coimmobilising glucose oxidase (GOD), fructose dehydrogenase (FDH), and the mediator, tetrathiafulvalene (TTF), by cross-linking with glutaraldehyde atop a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM) on a gold disk electrode (AuE). The performance of this bienzyme electrode under batch and flow injection (FI) conditions, as well as an amperometric detection in high-performance liquid chromatography (HPLC), are reported. The order of enzyme immobilisation atop the MPA-SAM affected the biosensor amperometric response in terms of sensitivity, with the immobilisation order GOD, FDH, TTF being selected. Similar analytical characteristics to those obtained with single GOD or FDH SAM-based biosensors for glucose and fructose were achieved with the bienzyme electrode, indicating that no noticeable changes in the biosensor responses to the analytes occurred as a consequence of the coimmobilisation of both enzymes on the same MPA–AuE. The suitability of the bienzyme biosensor for the analysis of real samples under flow injection conditions was tested by determining glucose in two certified serum samples. The simultaneous determination of glucose and fructose in the same sample cannot be performed without a separation step because at the detection potential used (+0.10 V), both sugars show amperometric response. Consequently, HPLC with amperometric detection at the TTF–FDH–GOD–MPA–AuE was accomplished. Glucose and fructose were simultaneously determined in honey, cola softdrink, and commercial apple juice, and the results were compared with those obtained by using other reference methods.

An integrated amperometric fructose biosensor based on a gold electrode (AuE) modified with a self-assembled monolayer (SAM) of 3-mercaptopropionic acid (MPA) on which fructose dehydrogenase (FDH) and the mediator tetrathiafulvalene (TTF) are co-immobilized by cross-linking with glutaraldehyde is reported. Variables concerning the behavior of the biosensor were optimized by taking the slope value obtained for the fructose calibration plot in the 0.1-1.0 mM concentration range as a criterion of selection. At an applied potential of +0.20 V, a good repeatability of such slope values (RSD=6.7%, n=10) was obtained with no need to apply a cleaning or pretreatment procedure to the modified electrode. Moreover, results from five different TTF-FDH-MPA-AuEs yielded a RSD of 5.8%. The useful lifetime of one single biosensor was approximately 30 days, exhibiting a 93% of the original response on the 33rd day. A linear calibration graph was obtained for fructose over the 1.0 x 10(-5)-1.0 x 10(-3) M range, with a limit of detection of 2.4 x 10(-6) M. The effect of potential interferents was evaluated. The TTF-FDH-MPA-AuE also performed well in the flow-injection mode. The biosensor was used for the determination of fructose in real samples, and the results compared with those provided by using a commercial enzyme test kit.

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.

The flow injection amperometric performance of solid composite graphite electrodes with ethylene/propylene/diene (EPD) or Teflon as binding agents, and with Ru or RuO2 particles as electrocatalytic modifiers has been compared. Both, Ru and RuO2 modified electrodes exhibited electrocatalytic properties on the methionine oxidation process in alkaline media. The electrodes composition and the hydrodynamic and chemical variables were optimized. Graphite-EPD (GEPD) electrodes showed a better analytical performance than graphite-Teflon (GPTFE) electrodes. Furthermore, a better sensitivity, repeatability and reproducibility was observed for RuO2-GEPD electrodes when compared with Ru-GEPD electrodes. At an applied potential of +0.50 V, a detection limit for methionine of 4.8x10(-5) mol L(-1), similar to those reported in the literature for other RuO2-modified electrodes, was obtained. The analytical applicability of RuO2-GEPD electrodes was demonstrated by determining methionine in a complex pharmaceutical formulation.

The possibility of applying adsorptive stripping voltammetry (AdSV) in dispersed media has been evaluated. Sensitive methods for the determination of the herbicide terbutryn at nanomolar levels by AdSV at the hanging mercury drop electrode in micellar and emulsified media are proposed. The anionic surfactant sodium pentanesulfonate was chosen as the most suitable agent for micellar solutions and oil–water emulsions. On the other hand, ethyl acetate was used as the organic solvent to form terbutryn emulsions. A systematic study of the various experimental parameters that affect the stripping response was carried out by differential pulse voltammetry. When working in a 0.1 mol L−l perchloric acid medium with an accumulation potential of −0.60 V, terbutryn could be determined over the 6.0 × 10−9−4.0 × 10−7 and 6.0 × 10−9−2.0 × 10−7 mol L−1 ranges in micellar (50s accumulation time) and emulsified (70s accumulation time) media, respectively. The limits of detection were 2.2 × 10−9 and 1.1 × 10−9 × mol L−1, and the relative standard deviations (n = 10) were 6.3 and 4.3% in micellar and emulsified media, respectively. The effect of other coexisting herbicides on the terbutryn stripping peak was evaluated. Finally, the developed method in emulsified medium was applied to the determination of terbutryn in spiked tap waters.

An electroanalytical study of the herbicide propazine's reduction process in micellar solutions and oil-in-water emulsions is reported. The anionic surfactant sodium pentanesulphonate was chosen as the most suitable. The differential pulse polarograms of micellar solutions had two reduction peaks below pH 2.0, whereas only one peak was obtained above pH 2.O. Ethyl acetate was chosen as the organic solvent to form propazine emulsions. Unlike in micellar solutions, the DPP polarograms of propazine emulsions showed only one peak even at pH < 2.0, suggesting that propazine hydrolysis was hindered in the emulsified medium. The limiting current is diffusion-controlled and the electrode process is irreversible. Propazine can be determined by differential pulse polarography over the 1.0 × 10−1 − 1.0 × 10−1moll−1 and 1.0 × 10−15 − 4.0 × 10−1 moll−1 concentration ranges and the limit of detection was 2.8 × 10−1 moll−1. Of the potential interferents simazine, methoprotryne and terbutryn (alls-triazines), thiram (a dithiocarbamate), dinoseb (nitrophenolic), and heptachlor (chlorinated cyclo-diene herbicide), only the first two were significant (10% error for equimolar concentrations). The method was applied to the determination of propazine in spiked drinking water. At a concentration level of 2.0 × 10−1 moll−1 a recovery of 94 ± 6% was obtained, after tenfold concentration on Sep-Pak.

An adsorptive stripping voltammetric method for the determination of the pesticide dinoseb (2-sec.-butyl-4,6-dinitrophenol) at the mercury film electrode is described. The deposition of the mercury film on a glassy carbon disk electrode was optimized. The temperature, at which the mercury film was deposited, was demonstrated to have a strong influence on the stripping peaks, the first one being much more intense than the second. A systematic study of the variables affecting the stripping response was carried out by differential pulse voltammetry. The results obtained have been compared with those at the HMDE; a significant improvement in the sensitivity of the method developed with the MFE was observed. Using a 300 s accumulation time, the limits of determination and detection were 3.6 × 10−10 and 1.1 × 10−10 mol L−1, respectively. The effect of the presence of several herbicides on the dinoseb response was also tested. The method has been applied to the determination of the pesticide in spiked apple juice at two concentration levels: 12.0 and 1.2 μg L−1 of juice.

Sensitive methods for the determination of the herbicides methoprotryne and terbutryn at nanomolar levels, by adsorptive stripping voltammetry at a hanging mercury drop electrode, are described. The electrocapillary curves, and cyclic voltammograms, demonstrate the adsorption of these compounds on the mercury electrode. A systematic study of the various experimental parameters that affect the stripping response was carried out by differential pulse voltammetry. The working medium chosen was 0.1 mol l–1 perchloric acid. By using an accumulation potential of –0.70 V and a 180 s accumulation time, the limits of detection were 5.2 × 10–10 and 2.4 × 10–9 mol l–1 for terbutryn and methoprotryne, respectively, and the relative standard deviations (n= 10) were 3.1% and 5.0% at concentration levels of 5.0 × 10–9 mol l–1 terbutryn and 1.0 × 10–8 mol l–1 methoprotryne, respectively. The degree of interference from some other herbicides on the differential pulse stripping signal for methoprotryne and terbutryn was evaluated. Finally, the methods were applied to the determination of terbutryn and methoprotryne in spiked irrigation and tap waters.

A sensitive method is described for the determination of Dinoseb (2-sec-butyl-4, 6-dinitrophenol) at nanomolar levels by adsorptive stripping voltammetry (AdSV) at the hanging mercury drop electrode. Cyclic voltammetry studies showed two well-defined reduction peaks, at −0.21 and −0.36 V, and demonstrated the adsorption of Dinoseb and its intermediate reduction product on the electrode. A systematic study of the experimental parameters that affect the stripping response was carried out using differential pulse voltammetry. Using a 120 second adsorption time, the limits of determination and detection were 1.5 × 10−9 and 4.6 × 10−10 M, respectively, and the relative standard deviation (n = 10) was 3.9% at a concentration level of 6.0 × 10−9 M.

An electroanalytical method for the determination of the herbicide desmetryne at nanomolar levels in dispersed media, based on adsorptive stripping voltammetry, is reported. The adsorption of desmetryne at the hanging mercury drop electrode was checked both in micellar solutions, where the anionic surfactant sodium pentanesulphonate was chosen as the most suitable surfactant agent, and in oil-in-water emulsions prepared with ethyl acetate as the organic solvent. In a micellar medium formed with 0.02% sodium pentanesulphonate and with 0.1 mol l −1 Britton–Robinson buffer (pH 1.5), the herbicide could be determined over the 1.0×10 −8 –4.0×10 −7 mol l −1 concentration range, when an accumulation potential of −0.70 V was applied for 50 s. On the other hand, in an oil-in-water emulsion formed with 2% ethyl acetate and 0.04% sodium pentanesulphonate as emulsifying agent in 0.1 mol l −1 HClO 4 , desmetryne could be determined over the 2.0×10 −9 –1.0×10 −7 mol l −1 concentration range. The limits of detection were 2.4×10 −9 and 4.2×10 −10 mol l −1 in micellar and emulsified media, respectively, with R.S.D.s ( n =10) 3.6 and 3.7%. The degree of interference from some other s-triazines on the desmetryne differential pulse response was also evaluated. Finally, the method developed in emulsified medium was applied to the determination of desmetryne in spiked apple juice.

Two flow injection (Fl) methods, conventional and stopped-flow, are described for the determination of formaldehyde based on its reaction with pararosaniline in the presence of sulphite ion followed by spectrophotometric detection at 570 nm. The calibration graphs are linear over the ranges 2.0–50 and 0.2–10 µg ml–1 of formaldehyde for conventional and stopped-flow Fl, respectively. The coefficient of variation was 6.3% at the 0.6 µg ml–1 level in the stopped-flow mode, the detection limit being 0.1 µg ml–1 of formaldehyde. Sampling rates of 41 and 18 samples h–1 were achieved in the conventional and stopped-flow modes, respectively, this being a major advantage of these methods. The effect of several organic and inorganic species was studied and only low relative molecular mass aldehydes were found to interfere. The stopped-flow method was applied to the determination of formaldehyde in air in work environments and its accuracy was confirmed by comparing the results with those obtained using the standard chromotropic acid method.