Angel Montoya

An integrated optical interferometric nanodevice based on silicon technology for biosensor applications

Integrated optical sensors have become important in recent years since they are the only technology which allows the direct detection of biomolecular interactions. Moreover, silicon microelectronics technology allows mass production as well as the fabrication of nano-/macrosystems on the same platform by hybrid integration of sources, sensors, photodetectors and complementary metal-oxide semiconductor electronics.For the fabrication of an optical sensor nanodevice with an integrated Mach–Zehnder interferometric (MZI) configuration, the optical waveguides must have two main features: monomode behaviour and a high surface sensitivity. In this paper we present the development of a MZI sensor based on total internal reflection waveguides with nanometre dimensions. The aim is to use these sensors in environmental control to detect water pollutants by immunoassay techniques.

Development of nanomechanical biosensors for detection of the pesticide DDT.

We report the use of a novel technique for detection of the organochlorine insecticide compound dichlorodiphenyltrichloroethane (DDT) by measuring the nanometer-scale bending of a microcantilever produced by differential surface stress. A synthetic hapten of the pesticide conjugated with bovine serum albumin (BSA) was covalently immobilised on the gold-coated side of the cantilever by using thiol self assembled monolayers. The immobilisation process is characterised by monitoring the cantilever deflection in real-time. Then specific detection is achieved by exposing the cantilever to a solution of a specific monoclonal antibody to the DDT hapten derivative. The specific binding of the antibodies on the cantilever sensitised side is measured with nanomolar sensitivity. Direct detection is proved by performing competitive assays, in which the cantilever is exposed to a mixed solution of the monoclonal antibody and DDT. The future prospects and limitations to be overcome for the application of nanomechanical sensors for pesticide detection are discussed.

Determination of environmental organic pollutants with a portable optical immunosensor

A portable surface plasmon resonance (SPR) optical biosensor device is described as a direct immunosensing system to determine organic pollutants in natural water samples. Monitoring of organochlorine (DDT), organophosphorus (chlorpyrifos) and carbamate (carbaryl) compounds within the concentration levels stipulated by the European legislation, can be accomplished using this immunosensor. The lowest limit of detection (LOD) was obtained for DDT, at 20 ng L(-1), whilst 50 ng L(-1) and 0.9 microg L(-1), were achieved for chlorpyrifos and carbaryl, respectively. Matrix effects were evaluated for the carbaryl immunoassay in different water types with detection limits within the range of carbaryl standard curves in distilled water (0.9-1.4 microg L(-1)). The covalent immobilization of the analyte derivative through an alkanethiol self-assembled monolayer (SAM) allowed the reusability of the sensor surface during more than 250 regeneration cycles. The quality of the regeneration was proved over a 1-month period of continuous working. The analysis time for a complete assay cycle, including regeneration, comprises 24 min. Our portable SPR-sensor system is already a market product, commercialized by the company SENSIA, SL. The size and electronic configuration of the device allow its portability and utilization on real contaminated locations.

Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo

SummaryHigh yields of human hepatocytes (up to 23×106 viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method. Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10−8M insulin, and 10−8M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177 nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was (3.50±0.17 nmol glucose·mg−1·min−1) similar to that reported for human liver. Insulin at 10−8M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10−9M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen, α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, haptoglobin, α2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by 10−9M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg−1 cell protein·min−1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver (35 nmol·mg−1).The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult human liver.

A piezoelectric immunosensor for the determination of pesticide residues and metabolites in fruit juices

A quartz crystal microbalance (QCM) immunosensor was developed for the determination of the insecticide carbaryl and 3,5,6-trichloro-2-pyridinol (TCP), the main metabolite of the insecticide chlorpyrifos and of the herbicide triclopyr. The detection was based on a competitive conjugate-immobilized immunoassay format using monoclonal antibodies (MAbs). Hapten conjugates were covalently immobilized, via thioctic acid self-assembled monolayer (SAM), onto the gold electrode sensitive surface of the quartz crystal. This covalent immobilization allowed the reusability of the modified electrode surface for at least one hundred and fifty assays without significant loss of sensitivity. The piezoimmunosensor showed detection limits (analyte concentrations producing 10% inhibition of the maximum signal) of 11 and 7 microg l(-1) for carbaryl and TCP, respectively. The sensitivity attained (I(50) value) was around 30 microg l(-1) for both compounds. Linear working ranges were 15-53 microg l(-1) for carbaryl and 13-83 microg l(-1) for TCP. Each complete assay cycle took 20 min. The good sensitivity, specificity, and reusability achieved, together with the short response time, allowed the application of this immunosensor to the determination of carbaryl and TCP in fruits and vegetables at European regulatory levels, with high precision and accuracy.

Determination of carbaryl, carbofuran and methiocarb in cucumbers and strawberries by monoclonal enzyme immunoassays and high-performance liquid chromatography with fluorescence detection. An analytical comparison.

Carbaryl, carbofuran and methiocarb are three of the most important N-methylcarbamate pesticides. In the present work, the application of laboratory-developed monoclonal antibody-based enzyme-linked immunosorbent assays (ELISAs) to the determination of these compounds in fruits and vegetables is described. Cucumbers and strawberries were spiked with the three carbamates at 10, 50 and 200 ppb. After extraction and clean-up, samples were analyzed by immunoassay and by HPLC with post-column derivatization and fluorescence detection (US Environmental Protection Agency Method 531.1). Results obtained by ELISA correlated well with those obtained by HPLC, both in terms of accuracy and precision. Recoveries were in the 60-90% range by ELISA and in the 50-90% range by HPLC, depending on the particular combination of commodity, pesticide, and fortification level under consideration. ELISAs were also applied to the analysis of non-purified sample extracts with excellent results: recoveries close to 100% were obtained, while maintaining similar precision values. This approach avoids the use of solid-phase extraction columns, saves time, and considerably increases the sample throughput. Results clearly indicate that the developed immunoassays may be suitable for the quantitative and reliable determination of carbaryl, carbofuran and methiocarb in fruits and vegetables even without including clean-up steps. These considerations make these ELISAs very useful analytical tools for monitoring and regulatory programs, without the need of complex and expensive instrumentation.

Development of a chemiluminescence-based quantitative lateral flow immunoassay for on-field detection of 2,4,6-trinitrotoluene

Simple, rapid and highly sensitive assays, possibly allowing on-site analysis, are required in the security and forensic fields or to obtain early signs of environmental pollution. Several bioanalytical methods and biosensors based on portable devices have been developed for this purpose. Among them, Lateral Flow ImmunoAssays (LFIAs) offer the advantages of rapidity and ease of use and, thanks to the high specificity of antigen-antibody binding, allow greatly simplifying and reducing sample pre-analytical treatments. However, LFIAs usually employ colloidal gold or latex beads as labels and they rely on the formation of colored bands visible by the naked eye. With this assay format, only qualitative or semi-quantitative information can be obtained and low sensitivity is achieved. Recently, the use of enzyme-catalyzed chemiluminescence detection in LFIA has been proposed to overcome these problems. In this work, we describe the development of a quantitative CL-LFIA assay for the detection of 2,4,6-trinitrotoluene (TNT) in real samples. Thanks to the use of a portable imaging device for CL signal measurement based on a thermoelectrically cooled CCD camera, the analysis could be performed directly on-field. A limit of detection of 0.2 μg mL(-1) TNT was obtained, which is five times lower than that obtained with a previously described colloidal gold-based LFIA developed employing the same immunoreagents. The dynamic range of the assay extended up to 5 μg mL(-1) TNT and recoveries ranging from 97% to 111% were obtained in the analysis of real samples (post blast residues obtained from controlled explosion).

Development of Monoclonal Immunoassays for the Determination of Triazole Fungicides in Fruit Juices

Enzyme-linked immunosorbent assays (ELISAs) based on monoclonal antibodies for the detection of triazole fungicides have been developed. With this aim, hapten-protein conjugates, containing the common triazole and chlorinated aromatic moieties, were prepared. From mice immunized with these conjugates, several monoclonal antibodies (MAbs) with the ability to sensitively bind several triazoles with different specificity were obtained. Both analyte- and class-specific ELISAs were developed. The hexaconazole-specific immunoassay can determine this fungicide with a limit of detection of 0.3 mug/L in standard buffer. The so-called triazole-specific immunoassay allowed for the detection of tetraconazole, penconazole, cyproconazole, and myclobutanil, with limits of detection in the 0.1-0.7 mug/L range. These immunoassays were applied to the determination of triazoles in spiked fruit juices. Samples were adequately diluted to minimize the matrix effects. Coefficients of variation were below 30%, and recoveries ranged from 62 to 135%. Therefore, the developed immunoassays can determine triazole fungicides in fruit juices down to the maximum residue limits currently legislated, without any sample treatment other than dilution.

A Comprehensive Overview on the Application of Flow Injection Techniques in Immunoanalysis

Abstract For more than a decade, immunoassay methods have held great promise for use not only in their classic clinical applications but also in food, pesticides, process and quality control, industrial fermentations, etc. A great number of publications have appeared in these fields. Flow injection analysis (FIA) is a technique that allows the automatic analysis of a large number of samples and/or continuous monitoring, and it has been proven to be applicable to a variety of analytical problems. Nevertheless, antibody-supported flow injection systems have received relatively little attention. Only recently, the advantage of the specificity inherent to most immune reactions began to be exploited in FIA procedures. Flow injection coupled to immunoassays (FIIA) is arising as a powerful tool for the development of analytical protocols, and the application of FIA techniques is expected to contribute enormously in improving the speed and quality of immunoassays. This article gives an overview on the application...

A monoclonal immunoassay for carbofuran and its application to the analysis of fruit juices

Abstract Monoclonal antibodies (MAbs) for the carbamate pesticide carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were obtained by immunizing mice with the hapten 3-[[(2,3-dihydro-2,2-dimethyl-7-benzofuranyloxy) carbonyl]amino] propanoic acid (BFNP) conjugated to bovine serum albumin (BSA). Based on one of these MAbs in combination with an heterologous hapten, an indirect enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of carbofuran. For standards, the detection limit of the ELISA was 0.2 ng ml−1, with a I50 value of 1.8 ng ml−1. Cross-reactivity studies showed that the immunoassay was quite specific for carbofuran since, of the six N-methylcarbamates assayed, only bendiocarb was significantly recognized (17.4%). The ELISA was applied to the determination of carbofuran in apple, grape, and pineapple juices. Recovery and precision of the method were evaluated by spiking juices with carbofuran at 25, 50, and 100 ng ml−1. Coefficients of variation were below 15% in most cases, and mean recoveries were 112.6, 107.4, and 115.4% for apple, grape, and pineapple juices, respectively. Therefore, the developed ELISA was able to determine carbofuran at levels below the maximum residue limits simply by diluting the sample, without the need for cleanup or sample concentration.