Maria-Pilar Marco

Biosensors for environmental monitoring: A global perspective

The intention of this article is to reflect the advances and describe the trends on biosensors for environmental applications. Biosensors are useful analytical tools for environmental monitoring, capable of providing results in real time, simple to use, portable and cost-effective. Some examples of biosensors in advanced stage of development, which have been applied to real samples, as well as of commercial devices, are given. Biosensors designed for measurement of either specific chemicals or their biological effects, such as toxicity biosensors and endocrine effect biosensors, are discussed. This overview also addresses the support provided by public institutions for biosensor research in the USA, Japan and, especially, in Europe. Future prospects of biosensor technology, with special emphasis in the development of new sensing elements, are foreseen.

Biosensors for environmental applications: Future development trends

Biosensors can be excellent analytical tools for monitoring programs working to implement legislation. In this article, biosensors for environmental analysis and monitoring are extensively reviewed. Examples of biosensors for the most important families of envi-ronmental pollutants, including some commercial devices, are presented. Finally, future trends in biosensor development are discussed. In this context, bioelectronics, nanotechnology, miniaturization, and especially biotechnology seem to be growing areas that will have a marked influence on the development of new biosensing strategies in the next future.

Environmental applications of analytical biosensors

A review of the fundamental aspects and environmental applications of biosensors is presented. The bases of different transducer principles such as electrochemical, optical and piezoelectric are discussed. Various examples are given of the applications of such principles to develop immunosensor devices to determine common environmental contaminants. Attention is also paid to catalytic biosensors, using enzymes as sensing elements. Biosensor devices based on the use of cholinesterase and various oxidase enzymes such as tyrosinase, laccase, peroxidase and aldehyde dehydrogenase are reported. Some examples are given of the applications of other biomolecules such as whole cells, DNA or proteins, to determine pollution. Validation studies are presented comparing biosensors with chromatographic techniques to determine organophosphorus pesticides and phenolic compounds in environmental samples.

Competitive flow immunoassay with fluorescence detection for determination of 4-nitrophenol

Abstract A selective competitive flow-immunoassay for monitoring of 4-nitrophenol, a demonstrated carcinogenic and mutagenic compound, is presented. The immunochemical detection principle is based on the competition between a fixed amount of labelled 4-nitrophenol derivative (tracer) and the analyte for a limited amount of antibody binding sites. The separation of free and bound tracer fractions is performed in a restricted-access (RA) column with subsequent fluorescence detection of the labelled immune complex. Optimisation of parameters, e.g. incubation time, flow rate, column size, type of antibody and tracer and their concentrations, was performed. The cross-reactivity for about 30 phenols, nitro-derivatives and other compounds was estimated. No major interferences except for 2,4-dinitrophenol and mercury(II) ions were observed. The optimised assay allowed a detection limit of 0.5xa0μgxa0l −1 4-nitrophenol (3.5xa0nM) with a sensitivity of 5.7xa0mg −1 xa0l, a sample throughput of 30 injections h −1 and a dynamic range between 5 and 1000xa0μgxa0l −1 4-nitrophenol. The assay was further tested on different sample matrices such as phosphate buffer, tap-, and rainwater without the need for any pre-treatment step.

Development of a highly sensitive enzyme-linked immunosorbent assay for atrazine Performance evaluation by flow injection immunoassay

Specific polyclonal antibodies to the herbicide atrazine (6-chloro-N-ethyl-N′-isopropyl-1,3,5-triazine-2,4-diamine) have been raised by immunizing three New Zealand rabbits. With the antisera (As) a highly sensitive enzyme-linked immunosorbent assays (ELISA) has been developed to determine atrazine in water samples. Several usable competitive immunoassays have been obtained by screening a battery of nine enzyme tracers (ETs) and three antisera. The optimized ELISA presents an IC50 of 0.28 nM (60 ng l−1) and a detection limit of 0.043 nM (9 ng l−1). Cross-reactivity studies have proved that the immunoassay is specific for atrazine while other triazine compounds are only detected on a minor extent. The flow injection immunoanalysis (FIIA) method has an IC50 of 2 nM (0.47 μg l−1) reaching a detection limit of 0.35 nM (75 ng l−1). The performance of both methods has been evaluated by analyzing water samples containing mixtures of atrazine and other pesticides at the ppb level. For this purpose two candidate reference materials have been used (A and B) and a spiked sample stored on Empore disks (sample C). A close correspondence was found between the results obtained with both immunochemical techniques.

Development and optimization of an indirect enzyme-linked immunosorbent assay for 4-nitrophenol. Application to the analysis of certified water samples

Abstract A polyclonal antibody-based enzyme-linked immunosorbent assay (ELISA) for the detection of 4-nitrophenol (4-NP) was developed, optimized and validated for the analysis of this polar compound in water samples. The concentrations of the immunoreagents were selected to provide the highest sensitivity on an indirect ELISA format. Moreover, the influence of several physico-chemical parameters, such as incubation time, ionic strength, detergent concentration and pH were also studied. The IC 50 and limit of detection (LOD) of the optimized assay As34/3-BSA (antiserum 34 and hapten 3 conjugated to bovine serum albumin) were 9.32 and 0.61xa0μgxa0l −1 , respectively. The specificity of this immunoassay was also evaluated by using eleven phenolic compounds, four different structurally related organophosphorus pesticides and a surfactant. Two kinds of sample matrices (Milli-Q and river water) were evaluated for possible interferences. A good correlation between the analysis by conventional techniques and by immunoassay was obtained, when five certified water samples were analyzed.

Preparation of antisera and development of a direct enzyme-linked immunosorbent assay for the determination of the antifouling agent Irgarol 1051

Abstract The development of an immunoassay to determine the antifouling agent Irgarol 1051 (2-methylthio-4- tert -butylamino-6-cyclopropylamino- s -triazine) in seawater samples is described. Three polyclonal antisera were obtained by immunizing with a hapten preserving important antigenic determinants such as the t -butyl and the methylthio groups of Irgarol. Protein conjugates were characterized by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Several usable competitive immunoassays have been obtained by screening a battery of 11 enzyme tracers. The optimized immunoassay presents an I 50 of 0.64 nM (0.163 μg/l) and a detection limit of 0.062 nM (0.016 μg/l). Cross-reactivity studies have demonstrated that the immunoassay is specific for Irgarol 1051. Other triazine compounds are only detected to a minor extent. Performance of the immunoassay is not affected by the high salinity content of seawater.

Validation of an immunoassay method for the determination of traces of carbaryl in vegetable and fruit extracts by liquid chromatography with photodiode array and mass spectrometric detection

A competitive enzyme-linked immunosorbent assay (ELISA) method for carbaryl quantitation in crop extracts was validated by liquid chromatography (LC) with diode array detection (DAD). For this purpose, six crops (banana, carrot, green bean, orange, peach and potato) were chosen for recovery and reproducibility studies. The general sample preparation included extraction with methanol followed by liquid-liquid partitioning and clean-up on Celite-charcoal adsorbent column of the vegetable extracts. ELISA samples consisted of a diluted LC extract in assay phosphate buffer (pH 7.5). The potential effect of methanol in these samples was evaluated. It was observed that a maximum content of 10% methanol present in the assay buffer could be tolerated without expressive losses in the ELISA performance. Under these conditions, a IC50 approximately 1.48 micrograms l-1 was obtained. A minimum matrix effect with a 1:50 dilution of the methanolic extracts in assay buffer was noticed, except for green bean samples that inhibited completely the assay. For the vegetable extracts, the ELISA sensitivities varied from 3.9 to 5.7 micrograms l-1, and good recoveries (82-96%) with R.S.D.s ranging from 5.7 to 12.1% were found. An excellent correlation between the LC-DAD and ELISA techniques was obtained. The confirmation of the carbaryl in less concentrated samples was achieved by LC-mass spectrometry interfaced with atmospheric pressure chemical ionisation. The [M + H]+ = 202 and [M + H-57]+ = 145 ions, equivalent to the protonated molecular and 1-naphthol ions, respectively, were used to carbaryl identification in these samples.

Development and application of immunoaffinity chromatography for the determination of the triazinic biocides in seawater

The development of an immunoaffinity chromatography (IAC) procedure for the selective extraction of the anti-fouling agent Irgarol 1051 [2-(tert.-butylamino)-4-(cyclopropylamino)-6-(methylthio)-1,3,5-triazine] from seawater is described. The anti-Irgarol 1051 antibodies were covalently bound to agarose-based beads support. IAC column capacities were higher than 400 ng and ethanol-water (70:30) was selected as eluting mixture. After percolation of 250 ml of water sample containing Irgarol 1051 at environmental levels (ng l(-1) ), the breakthrough volume was still not achieved. Other triazine herbicides percolated through the IAC column showed good recoveries. Thus, this IAC procedure may be useful to extract related compounds. The developed IAC column was applied to real seawater samples and compared with RP-C18 cartridges. The limit of detection (LOD) reached by using the IAC procedure was twenty times lower than the LOD achieved by the RP-C 18 cartridges using the same detection system. Irgarol 1051 was detected at ng l(-1) levels in the Barcelona marina (northwestern Mediterranean Sea). An acceptable correlation between enzyme-linked immunosorbent assay and gas chromatography with nitrogen-phosphorus detection was observed, thus analysis of Irgarol 1051 can be performed by either one of the methods. In this work, further confirmation of the analyte identity for real samples was accomplished by gas chromatography-electron impact mass spectrometry.

Effect of competitor design on immunoassay specificity: Development and evaluation of an enzyme-linked immunosorbent assay for 2,4-dinitrophenol

Abstract The specificity of an enzyme-linked immunosorbent assay (ELISA) has been modified by changing the chemical structure of the haptenized coating antigen. A competitive indirect immunoassay has been developed, evaluated and validated to measure 2,4-dinitrophenol (2,4-DNP) in wastewater samples. The assay uses polyclonal antisera originally produced to analyze 4-nitrophenol (4-NP), but changing the chemical structure of the competitor, the resulting immunoassay is able to detect specifically 2,4-DNP in the presence of 4-NP. The IC 50 of the optimized immunoassay As36/4-OVA (antiserum 36 and hapten 4 attached to ovalbumin as coating antigen) is 2.03xa0μgxa0l −1 and the limit of detection (LOD) 0.26xa0μgxa0l −1 . The working range of the assay is placed between 0.55 and 7.32xa0μgxa0l −1 . Moreover, the influence of several physico-chemical parameters such as incubation time, ionic strength, detergent concentration and pH were studied. The specificity of this immunoassay was evaluated using 35 structurally related compounds. Two kinds of water matrices (Milli-Q and river water) have been used to study possible interferences and to establish the accuracy and precision of the assay. Finally, a good correlation was observed between the chromatographic and immunoassay techniques while analyzing five certified water samples.