The potential of thiol-ene chemistry as a selective strategy to functionalize silicon materials f... more The potential of thiol-ene chemistry as a selective strategy to functionalize silicon materials for DNA microarraying is demonstrated and applied to discriminate genetic variations.
A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to... more A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15 ng/g, reaching qualitative results within 10 min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n = 25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.
Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that m... more Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications. However, aluminum has been scarcely used for biosensing purposes due to corrosion and pitting issues. In this short review, we show our recent achievements on aluminum nanohole platforms for (bio)sensing. These include a method to circumvent aluminum degradation-which has been successfully applied to the demonstration of
Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from... more Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from conventional compact discs (CD). Standard micro and nanofabrication processes have been used and optimized to be PC compatible. The viability of this CD-based plasmonic platform for labelfree optical biosensing has been demonstrated through a competitive bioassay for biotin analysis using biotinfunctionalized dextran-lipase conjugates immobilized on the transducer surface.
► High prevalence of intersex gonads was found in the Urdaibai Biosphere Reserve. ► High levels o... more ► High prevalence of intersex gonads was found in the Urdaibai Biosphere Reserve. ► High levels of alkylphenols were measured in fish bile. ► VTG protein was detected in the plasma of undifferentiated, male and intersex fish. ► Levels of phthalate and organotins measured in sediments are of great concern. ► This study provided evidence of EDs presence in the Urdaibai Biosphere Reserve. a b s t r a c t
Summary The effect of one application of the slow-release fertilizer, sulfur-coated urea, on the ... more Summary The effect of one application of the slow-release fertilizer, sulfur-coated urea, on the nitrogen level in leaves, fruit and bark of Washington Navel orange trees was studied during the different periods of the annual vegetative cycle, in comparison with two annual applications of the fertilizer ammonium nitrosulphate.
Critical Reviews in Food Science and Nutrition, 1999
Detection of pesticides and their metabolites in food and environmental samples in real time is t... more Detection of pesticides and their metabolites in food and environmental samples in real time is the goal of many industries. Immunoassay technology has several attributes that make it a useful tool for screening purposes (e.g., selectivity, sensitivity, portability, and rapid turnaround time). Approximately 90% of the developed immunoassays for the pesticide residue analysis use the ELISA technique. Commercial kits are tailored to target different analytes, thus eliminating in some cases the need for clean-up steps. The manageability of the immunoassay test kit, together with its accuracy and speed of analysis, allows the rapid determination in situ of many samples simultaneously. This article gives an overview on the applications of the immunokits for pesticide analysis in drinking water and foods, as well as examples of different immunoassay formats commonly used. Special attention is given to sample extraction and clean-up procedures. Application to the determination of common pesticides and their detection limit are summarized. Immunoassay kits offer many practical advantages, and the acceptance of these methods depends on several factors, including the demonstration of quality and validity compared with reference methods. Although the advantages of the technique and their applications to food industry quality control are scarcely referred to in the literature.
The selective introduction of functional groups on the surface of silicon nitride/silicon oxide n... more The selective introduction of functional groups on the surface of silicon nitride/silicon oxide nanostructures was studied. Chemical strategies based on organosilane, Si-H and N-H reactivities were assayed. Among these strategies, the use of glutaraldehyde to selectively immobilize biomolecules only on the silicon nitride part of the chip surface was the most effective for the covalent attachment of proteins, maintaining also their bioavailability. The biomolecule surface coverage results up to 80% and the modification is selective versus silicon oxide; the biomolecule attaching only to silicon nitride and leaving the silicon oxide area of the device unmodified. The effectiveness of our novel selective surface modification procedure is also supported by comparing experimental and numerical calculations of the optical performance of a label-free optical ring resonator based on Si 3 N 4 /SiO 2 slot-waveguides.
Immunosensor systems have been developed for the rapid determination of l-naphthol. In this work,... more Immunosensor systems have been developed for the rapid determination of l-naphthol. In this work, the comparison of performance of immunosensors working in aqueous and organic media was done. Direct, indirect and capture formats were studied. Immunoreagents were immobilized on controlled pore glass (CPG), hidroxysuccinimide agarose gel or on azlactone Protein A/G supports. The Protein A/G-based sensor showed the best performance. In aqueous media, a LOD of 16.2 mg l − 1 and a DR of 33.7-586.6 mg l − 1 were achieved employing Tween 20 at a concentration ranging from 0.01 to 0.05% v/v. Maximum sensitivity was reached with 0.025% of surfactant. Binary mixtures of methanol or acetonitrile with aqueous buffer and ternary mixtures of methanol/isopropanol or ethyl acetate/methanol with the same buffer were studied as organic media. The mixture 50% MeOH-50% 20 mM sodium phosphate, pH 8, with 0.05% (v/v) Tween 20 resulted to be the best. A detection limit of 12.0 mg l − 1 and a dynamic range of 53.6-17 756.0 mg l − 1 were reached. The recycling of Protein A/G-based sensor working in this media was about 300 assays. Preconcentration factors around 250 were achieved using methanol as extracting solvent. It has been demonstrated that the technique can be successful in carrying out the analysis of low solubility in water analytes, such as l-naphthol. The sensors developed can use higher concentrations of organic solvent (up to 50% methanol) compared to ELISA. On the other hand, the advantage of preconcentration can also be taken for the use of the same procedure as recommended for standard sample treatments.
In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to perio... more In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to periodic networks of nano-pillar suitable for biosensing when are vertically interrogated. In this article, we demonstrate the biosensing capabilities of a type of micrometric size BICELLs made of SU-8 nano-pillars fabricated over transparent substrates. We verify the biochips functionality comparing the theoretical simulations with the experimental results when are optically interrogated in transmission. We also demonstrate a sensitivity enhancement by reducing the pitch among nano-pillars from 800 to 700 nm. Thus, the Limit of Detection achievable in these types of BICELLs is in the order of 64 pg/mL for 700 nm in pitch among nano-pillars in comparison with 292 pg/mL for 800 nm in pitch when are interrogated by Fourier Transform Visible and Infrared Spectrometry. The experiments exhibited a good reproducibility with a relative standard deviation of 0.29% measured within 8 days for a specific concentration.
Selected physico-chemical and chemical characteristics of 13 coconut coir dust (mesocarp pithy ti... more Selected physico-chemical and chemical characteristics of 13 coconut coir dust (mesocarp pithy tissue plus short-length ®bres) samples from Asia, America and Africa were evaluated as peat alternatives. All properties studied diered signi®cantly between and within sources, and from the control Sphagnum peat. pH of coir dust was slightly acidic, whereas salinity varied dramatically between 39 and 597 mS m À1 in the saturated media extract. The cation exchange capacity and carbon/nitrogen (C/N) ratio ranged from 31.7 to 95.4 cmol c kg À1 and from 75 to 186, respectively. Most carbon was found as lignin and cellulose. The concentrations of available nitrogen, calcium, magnesium and micro-elements were low, while those of phosphorus and potassium were remarkably high (0.28±2.81 mol m À3 and 2.97±52.66 mol m À3 for P and K, respectively). Saline ion concentrations, especially chloride and sodium, were also high. Ó
Antibiotic residues in edible animal products are of great concern to regulatory agencies and con... more Antibiotic residues in edible animal products are of great concern to regulatory agencies and consumers, so reliable screening methods for rapid, selective and sensitive detection of these residues are necessary to ensure food safety.
The development of simple, accurate, and rapid multisample analytical methodologies to find out c... more The development of simple, accurate, and rapid multisample analytical methodologies to find out critical targets in waters is highly demanded. Optical microsensor arrays to determine microcystins in river waters are developed on the polycarbonate side of compact discs. The working principle of the sensors relied on an indirect competitive microimmunoassay, where free microcystin LR (MC-LR) competes with immobilized conjugate for specific monoclonal antibody. The results of the immunoreaction are detected with a DVD drive, showing the readouts in minutes. The method reached a sensitivity (IC 50 ) for MC-LR of 1.04 µg/L and a linear response in the range 0.12-2.00 µg/ L, allowing its determination below the upper limit proposed by the World Health Organization in drinking water. The developed analytical approach shows simplicity, good sensitivity, high throughput capability, and rapidity (37 min) in field use. The optimized assay showed also high congener reactivity to MC-LY (144%), MC-LA (125%), MC-LF (119%), MC-LW (102%), MC-YR (83%), and nodularin (94%). Furthermore, the suitability of the disk biosensor to quantify MC-LR was successfully evaluated analyzing river water samples, obtaining excellent recoveries (78-113%). Precoated discs are stable for at least seven weeks without loosing their analytical performances. Also, the portability of the analytical system permits on-site analysis and quantification, saving time and other resources. To our knowledge, this is the only work where a portable, easy-touse, array based system has been developed for on-site microcystin quantification and applied to simultaneously analyze 42 samples plus the calibration curve, reaching microgram per liter sensitivity.
We developed biophotonic sensing arrays of 60 × 60 m 2 made of periodic lattices of high aspect r... more We developed biophotonic sensing arrays of 60 × 60 m 2 made of periodic lattices of high aspect ratio SU-8 nano-pillars in order to demonstrate their capability for label-free molecule detection, as well as the sensitivity enhancement in comparison with a single layer of SU-8. The biophotonic sensing arrays, that we call BICELLs (Biophotonic sensing cells), are interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR). We monitored the surface immobilization of bovine serum albumin (BSA) antigen and anti-BSA antibody (aBSA) recognition. The bioassay exhibits a limit of detection (LOD) in the order of 2 ng/ml limited by the wavenumber uncertainty during the interrogation process. We also estimated and compared the theoretical biolayer thickness with previous results.
The potential of thiol-ene chemistry as a selective strategy to functionalize silicon materials f... more The potential of thiol-ene chemistry as a selective strategy to functionalize silicon materials for DNA microarraying is demonstrated and applied to discriminate genetic variations.
A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to... more A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15 ng/g, reaching qualitative results within 10 min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n = 25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC-MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.
Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that m... more Sub-wavelength diameter holes in thin metal layers can exhibit remarkable optical features that make them highly suitable for (bio)sensing applications. Either as efficient light scattering centers for surface plasmon excitation or metal-clad optical waveguides, they are able to form strongly localized optical fields that can effectively interact with biomolecules and/or nanoparticles on the nanoscale. As the metal of choice, aluminum exhibits good optical and electrical properties, is easy to manufacture and process and, unlike gold and silver, its low cost makes it very promising for commercial applications. However, aluminum has been scarcely used for biosensing purposes due to corrosion and pitting issues. In this short review, we show our recent achievements on aluminum nanohole platforms for (bio)sensing. These include a method to circumvent aluminum degradation-which has been successfully applied to the demonstration of
Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from... more Al nanohole array plasmonic biosensors have been fabricated on polycarbonate (PC) substrates from conventional compact discs (CD). Standard micro and nanofabrication processes have been used and optimized to be PC compatible. The viability of this CD-based plasmonic platform for labelfree optical biosensing has been demonstrated through a competitive bioassay for biotin analysis using biotinfunctionalized dextran-lipase conjugates immobilized on the transducer surface.
► High prevalence of intersex gonads was found in the Urdaibai Biosphere Reserve. ► High levels o... more ► High prevalence of intersex gonads was found in the Urdaibai Biosphere Reserve. ► High levels of alkylphenols were measured in fish bile. ► VTG protein was detected in the plasma of undifferentiated, male and intersex fish. ► Levels of phthalate and organotins measured in sediments are of great concern. ► This study provided evidence of EDs presence in the Urdaibai Biosphere Reserve. a b s t r a c t
Summary The effect of one application of the slow-release fertilizer, sulfur-coated urea, on the ... more Summary The effect of one application of the slow-release fertilizer, sulfur-coated urea, on the nitrogen level in leaves, fruit and bark of Washington Navel orange trees was studied during the different periods of the annual vegetative cycle, in comparison with two annual applications of the fertilizer ammonium nitrosulphate.
Critical Reviews in Food Science and Nutrition, 1999
Detection of pesticides and their metabolites in food and environmental samples in real time is t... more Detection of pesticides and their metabolites in food and environmental samples in real time is the goal of many industries. Immunoassay technology has several attributes that make it a useful tool for screening purposes (e.g., selectivity, sensitivity, portability, and rapid turnaround time). Approximately 90% of the developed immunoassays for the pesticide residue analysis use the ELISA technique. Commercial kits are tailored to target different analytes, thus eliminating in some cases the need for clean-up steps. The manageability of the immunoassay test kit, together with its accuracy and speed of analysis, allows the rapid determination in situ of many samples simultaneously. This article gives an overview on the applications of the immunokits for pesticide analysis in drinking water and foods, as well as examples of different immunoassay formats commonly used. Special attention is given to sample extraction and clean-up procedures. Application to the determination of common pesticides and their detection limit are summarized. Immunoassay kits offer many practical advantages, and the acceptance of these methods depends on several factors, including the demonstration of quality and validity compared with reference methods. Although the advantages of the technique and their applications to food industry quality control are scarcely referred to in the literature.
The selective introduction of functional groups on the surface of silicon nitride/silicon oxide n... more The selective introduction of functional groups on the surface of silicon nitride/silicon oxide nanostructures was studied. Chemical strategies based on organosilane, Si-H and N-H reactivities were assayed. Among these strategies, the use of glutaraldehyde to selectively immobilize biomolecules only on the silicon nitride part of the chip surface was the most effective for the covalent attachment of proteins, maintaining also their bioavailability. The biomolecule surface coverage results up to 80% and the modification is selective versus silicon oxide; the biomolecule attaching only to silicon nitride and leaving the silicon oxide area of the device unmodified. The effectiveness of our novel selective surface modification procedure is also supported by comparing experimental and numerical calculations of the optical performance of a label-free optical ring resonator based on Si 3 N 4 /SiO 2 slot-waveguides.
Immunosensor systems have been developed for the rapid determination of l-naphthol. In this work,... more Immunosensor systems have been developed for the rapid determination of l-naphthol. In this work, the comparison of performance of immunosensors working in aqueous and organic media was done. Direct, indirect and capture formats were studied. Immunoreagents were immobilized on controlled pore glass (CPG), hidroxysuccinimide agarose gel or on azlactone Protein A/G supports. The Protein A/G-based sensor showed the best performance. In aqueous media, a LOD of 16.2 mg l − 1 and a DR of 33.7-586.6 mg l − 1 were achieved employing Tween 20 at a concentration ranging from 0.01 to 0.05% v/v. Maximum sensitivity was reached with 0.025% of surfactant. Binary mixtures of methanol or acetonitrile with aqueous buffer and ternary mixtures of methanol/isopropanol or ethyl acetate/methanol with the same buffer were studied as organic media. The mixture 50% MeOH-50% 20 mM sodium phosphate, pH 8, with 0.05% (v/v) Tween 20 resulted to be the best. A detection limit of 12.0 mg l − 1 and a dynamic range of 53.6-17 756.0 mg l − 1 were reached. The recycling of Protein A/G-based sensor working in this media was about 300 assays. Preconcentration factors around 250 were achieved using methanol as extracting solvent. It has been demonstrated that the technique can be successful in carrying out the analysis of low solubility in water analytes, such as l-naphthol. The sensors developed can use higher concentrations of organic solvent (up to 50% methanol) compared to ELISA. On the other hand, the advantage of preconcentration can also be taken for the use of the same procedure as recommended for standard sample treatments.
In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to perio... more In a previous work we introduced the term Bio-Photonic Sensing Cells (BICELLs), referred to periodic networks of nano-pillar suitable for biosensing when are vertically interrogated. In this article, we demonstrate the biosensing capabilities of a type of micrometric size BICELLs made of SU-8 nano-pillars fabricated over transparent substrates. We verify the biochips functionality comparing the theoretical simulations with the experimental results when are optically interrogated in transmission. We also demonstrate a sensitivity enhancement by reducing the pitch among nano-pillars from 800 to 700 nm. Thus, the Limit of Detection achievable in these types of BICELLs is in the order of 64 pg/mL for 700 nm in pitch among nano-pillars in comparison with 292 pg/mL for 800 nm in pitch when are interrogated by Fourier Transform Visible and Infrared Spectrometry. The experiments exhibited a good reproducibility with a relative standard deviation of 0.29% measured within 8 days for a specific concentration.
Selected physico-chemical and chemical characteristics of 13 coconut coir dust (mesocarp pithy ti... more Selected physico-chemical and chemical characteristics of 13 coconut coir dust (mesocarp pithy tissue plus short-length ®bres) samples from Asia, America and Africa were evaluated as peat alternatives. All properties studied diered signi®cantly between and within sources, and from the control Sphagnum peat. pH of coir dust was slightly acidic, whereas salinity varied dramatically between 39 and 597 mS m À1 in the saturated media extract. The cation exchange capacity and carbon/nitrogen (C/N) ratio ranged from 31.7 to 95.4 cmol c kg À1 and from 75 to 186, respectively. Most carbon was found as lignin and cellulose. The concentrations of available nitrogen, calcium, magnesium and micro-elements were low, while those of phosphorus and potassium were remarkably high (0.28±2.81 mol m À3 and 2.97±52.66 mol m À3 for P and K, respectively). Saline ion concentrations, especially chloride and sodium, were also high. Ó
Antibiotic residues in edible animal products are of great concern to regulatory agencies and con... more Antibiotic residues in edible animal products are of great concern to regulatory agencies and consumers, so reliable screening methods for rapid, selective and sensitive detection of these residues are necessary to ensure food safety.
The development of simple, accurate, and rapid multisample analytical methodologies to find out c... more The development of simple, accurate, and rapid multisample analytical methodologies to find out critical targets in waters is highly demanded. Optical microsensor arrays to determine microcystins in river waters are developed on the polycarbonate side of compact discs. The working principle of the sensors relied on an indirect competitive microimmunoassay, where free microcystin LR (MC-LR) competes with immobilized conjugate for specific monoclonal antibody. The results of the immunoreaction are detected with a DVD drive, showing the readouts in minutes. The method reached a sensitivity (IC 50 ) for MC-LR of 1.04 µg/L and a linear response in the range 0.12-2.00 µg/ L, allowing its determination below the upper limit proposed by the World Health Organization in drinking water. The developed analytical approach shows simplicity, good sensitivity, high throughput capability, and rapidity (37 min) in field use. The optimized assay showed also high congener reactivity to MC-LY (144%), MC-LA (125%), MC-LF (119%), MC-LW (102%), MC-YR (83%), and nodularin (94%). Furthermore, the suitability of the disk biosensor to quantify MC-LR was successfully evaluated analyzing river water samples, obtaining excellent recoveries (78-113%). Precoated discs are stable for at least seven weeks without loosing their analytical performances. Also, the portability of the analytical system permits on-site analysis and quantification, saving time and other resources. To our knowledge, this is the only work where a portable, easy-touse, array based system has been developed for on-site microcystin quantification and applied to simultaneously analyze 42 samples plus the calibration curve, reaching microgram per liter sensitivity.
We developed biophotonic sensing arrays of 60 × 60 m 2 made of periodic lattices of high aspect r... more We developed biophotonic sensing arrays of 60 × 60 m 2 made of periodic lattices of high aspect ratio SU-8 nano-pillars in order to demonstrate their capability for label-free molecule detection, as well as the sensitivity enhancement in comparison with a single layer of SU-8. The biophotonic sensing arrays, that we call BICELLs (Biophotonic sensing cells), are interrogated vertically by using micron spot size Fourier transform visible and IR spectrometry (FT-VIS-IR). We monitored the surface immobilization of bovine serum albumin (BSA) antigen and anti-BSA antibody (aBSA) recognition. The bioassay exhibits a limit of detection (LOD) in the order of 2 ng/ml limited by the wavenumber uncertainty during the interrogation process. We also estimated and compared the theoretical biolayer thickness with previous results.
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Papers by R. Puchades