45 results on '"Santiago-Felipe, Sara"'
Search Results
2. A fluorogenic capped mesoporous aptasensor for gluten detection
- Author
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Pla, Luis, Martínez-Bisbal, M. Carmen, Aznar, Elena, Sancenón, Félix, Martínez-Máñez, Ramón, and Santiago-Felipe, Sara
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- 2021
- Full Text
- View/download PDF
3. Aptamer-Capped nanoporous anodic alumina for Staphylococcus aureus detection
- Author
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Pla, Luis, Santiago-Felipe, Sara, Tormo-Mas, María Ángeles, Pemán, Javier, Sancenón, Félix, Aznar, Elena, and Martínez-Máñez, Ramón
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- 2020
- Full Text
- View/download PDF
4. Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation
- Author
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Ribes, Àngela, Santiago-Felipe, Sara, Aviñó, Anna, Candela-Noguera, Vicente, Eritja, Ramón, Sancenón, Félix, Martínez-Máñez, Ramón, and Aznar, Elena
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- 2018
- Full Text
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5. Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors
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Santiago-Felipe, Sara, Tortajada-Genaro, Luis A., Carrascosa, Javier, Puchades, Rosa, and Maquieira, Ángel
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- 2016
- Full Text
- View/download PDF
6. Aptamer-capped nanoporous anodic alumina for SARS-CoV-2 spike protein detection
- Author
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Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Ciencia e Innovación (España), European Commission, Agencia Estatal de Investigación (España), Generalitat Valenciana, Instituto de Investigación Sanitaria La Fe (España), Universidad Politécnica de Valencia, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Caballos, Isabel, Aranda, María Nieves, López-Palacios, Alba, Pla, Luis, Santiago-Felipe, Sara, Hernández-Montoto, Andy, Tormo-Mas, María Ángeles, Pemán, Javier, Gómez-Ruiz, María Dolores, Calabuig, Eva, Sánchez-Sendra, Beatriz, Francés-Gómez, Clara, Geller, Ron, Aznar, Elena, Martínez-Máñez, Ramón, Ministerio de Ciencia, Innovación y Universidades (España), Ministerio de Ciencia e Innovación (España), European Commission, Agencia Estatal de Investigación (España), Generalitat Valenciana, Instituto de Investigación Sanitaria La Fe (España), Universidad Politécnica de Valencia, Ministerio de Economía y Competitividad (España), Instituto de Salud Carlos III, Caballos, Isabel, Aranda, María Nieves, López-Palacios, Alba, Pla, Luis, Santiago-Felipe, Sara, Hernández-Montoto, Andy, Tormo-Mas, María Ángeles, Pemán, Javier, Gómez-Ruiz, María Dolores, Calabuig, Eva, Sánchez-Sendra, Beatriz, Francés-Gómez, Clara, Geller, Ron, Aznar, Elena, and Martínez-Máñez, Ramón
- Abstract
The COVID-19 pandemic, which began in 2019, has highlighted the importance of testing and tracking infected individuals as a means of mitigating the spread of the virus. In this context, the development of sensitive and rapid methods for the detection of SARS-CoV-2, the virus responsible for COVID-19, is crucial. Herein, a biosensor based on oligonucleotide-gated nanomaterials for the specific detection of SARS-CoV-2 spike protein is presented. The sensing system consists of a nanoporous anodic alumina disk loaded with the fluorescent indicator rhodamine B and capped with a DNA aptamer that selectively binds the SARS-CoV-2 spike protein. The system is initially evaluated using pseudotype virus systems based on vesicular stomatitis virus carrying different SARS-CoV-2 S-proteins on their surface. When the pseudotype virus is present, the cap of the solid is selectively removed, triggering the release of the dye from the pore voids to the medium. The nanodevice demonstrated its ability to detect pseudotype virus concentrations as low as 7.5·103 PFU mL. In addition, the nanodevice is tested on nasopharyngeal samples from individuals suspected of having COVID-19.
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- 2023
7. Aptamer‐Capped Nanoporous Anodic Alumina for SARS‐CoV‐2 Spike Protein Detection
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Caballos, Isabel, primary, Aranda, María Nieves, additional, López‐Palacios, Alba, additional, Pla, Luis, additional, Santiago‐Felipe, Sara, additional, Hernández‐Montoto, Andy, additional, Tormo‐Mas, María Ángeles, additional, Pemán, Javier, additional, Gómez‐Ruiz, María Dolores, additional, Calabuig, Eva, additional, Sánchez‐Sendra, Beatriz, additional, Francés‐Gómez, Clara, additional, Geller, Ron, additional, Aznar, Elena, additional, and Martínez‐Máñez, Ramón, additional
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- 2023
- Full Text
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8. One-pot isothermal DNA amplification – Hybridisation and detection by a disc-based method
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Santiago-Felipe, Sara, Tortajada-Genaro, Luis A., Morais, Sergi, Puchades, Rosa, and Maquieira, Ángel
- Published
- 2014
- Full Text
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9. Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc
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Santiago-Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, and Maquieira, Ángel
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- 2016
- Full Text
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10. A fluorogenic capped mesoporous aptasensor for gluten detection
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Universitat Politècnica de València. Instituto de Reconocimiento Molecular y Desarrollo Tecnológico - Institut de Reconeixement Molecular i Desenvolupament Tecnològic, GENERALITAT VALENCIANA, Instituto de Salud Carlos III, AGENCIA ESTATAL DE INVESTIGACION, Universitat Politècnica de València, Pla, Luis, Martínez-Bisbal, M.Carmen, Aznar, Elena, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, Santiago Felipe, Sara, Universitat Politècnica de València. Departamento de Química - Departament de Química, Universitat Politècnica de València. Instituto de Reconocimiento Molecular y Desarrollo Tecnológico - Institut de Reconeixement Molecular i Desenvolupament Tecnològic, GENERALITAT VALENCIANA, Instituto de Salud Carlos III, AGENCIA ESTATAL DE INVESTIGACION, Universitat Politècnica de València, Pla, Luis, Martínez-Bisbal, M.Carmen, Aznar, Elena, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, and Santiago Felipe, Sara
- Abstract
[EN] Celiac disease is a complex and autoimmune disorder caused by the ingestion of gluten affecting almost 1% of global population. Nowadays an effective treatment does not exist, and the only way to manage the disease is the removal of gluten from the diet. Owing the key role played by gluten, clear and regulated labelling of foodstuff and smart methods for gluten detection are needed to fight frauds on food industry and to avoid the involuntary ingestion of this protein by celiac patients. On that scope, the development of a novel detection system of gluten is here presented. The sensor consists of nanoporous anodic alumina films loaded with a fluorescent dye and capped with an aptamer that recognizes gliadin (gluten's soluble proteins). In the presence of gliadin, aptamer sequences displace from the surface of anodic alumina resulting in pore opening and dye delivery. The dispositive shows a limit of detection (LOD) of 100 mu g kg(-1) of gliadin, good selectivity and a detection time of approximately 60 min. Moreover, the sensor is validated in real food samples. This novel probe allows fast gluten detection through a simple signalling process with potential use for food control. (C) 2020 Elsevier B.V. All rights reserved.
- Published
- 2021
11. Nanoporous Anodic Alumina-Based Sensor for miR-99a-5p Detection as an Effective Early Breast Cancer Diagnostic Tool
- Author
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Garrido-Cano, Iris, primary, Pla, Luis, additional, Santiago-Felipe, Sara, additional, Simón, Soraya, additional, Ortega, Belen, additional, Bermejo, Begoña, additional, Lluch, Ana, additional, Cejalvo, Juan Miguel, additional, Eroles, Pilar, additional, and Martínez-Máñez, Ramón, additional
- Published
- 2021
- Full Text
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12. A new 8-oxo-7,8-2′deoxyguanosine nanoporous anodic alumina aptasensor for colorectal cancer diagnosis in blood and urine
- Author
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Pla, Luis, primary, Sancenón, Félix, additional, Martínez-Bisbal, M. Carmen, additional, Bañuls, Celia, additional, Estañ, Nuria, additional, Botello-Marabotto, Marina, additional, Aznar, Elena, additional, Sáez, Guillermo, additional, Santiago-Felipe, Sara, additional, and Martínez-Máñez, Ramón, additional
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- 2021
- Full Text
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13. Oligonucleotide-capped nanoporous anodic alumina biosensor as diagnostic tool for rapid and accurate detection of Candida auris in clinical samples
- Author
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Pla, Luis, primary, Santiago-Felipe, Sara, additional, Tormo-Mas, María Ángeles, additional, Ruiz-Gaitán, Alba, additional, Pemán, Javier, additional, Valentín, Eulogio, additional, Sancenón, Félix, additional, Aznar, Elena, additional, and Martínez-Máñez, Ramón, additional
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- 2021
- Full Text
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14. Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples
- Author
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Pla, Luis, primary, Aviñó, Anna, additional, Eritja, Ramón, additional, Ruiz-Gaitán, Alba, additional, Pemán, Javier, additional, Friaza, Vicente, additional, Calderón, Enrique J., additional, Aznar, Elena, additional, Martínez-Máñez, Ramón, additional, and Santiago-Felipe, Sara, additional
- Published
- 2020
- Full Text
- View/download PDF
15. Aptamer-Capped nanoporous anodic alumina for Staphylococcus aureus detection
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, European Social Fund, Generalitat Valenciana, Instituto de Salud Carlos III, Agencia Estatal de Investigación, European Regional Development Fund, Universitat Politècnica de València, Instituto de Investigación Sanitaria La Fe, Ministerio de Ciencia, Innovación y Universidades, Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Pla, Luis, Santiago Felipe, Sara, Tormo-Mas, María Ángeles, Pemán, Javier, Sancenón Galarza, Félix, Aznar, Elena, Martínez-Máñez, Ramón, Universitat Politècnica de València. Departamento de Química - Departament de Química, European Social Fund, Generalitat Valenciana, Instituto de Salud Carlos III, Agencia Estatal de Investigación, European Regional Development Fund, Universitat Politècnica de València, Instituto de Investigación Sanitaria La Fe, Ministerio de Ciencia, Innovación y Universidades, Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Pla, Luis, Santiago Felipe, Sara, Tormo-Mas, María Ángeles, Pemán, Javier, Sancenón Galarza, Félix, Aznar, Elena, and Martínez-Máñez, Ramón
- Abstract
[EN] The development of new detection systems for an accurate and rapid identification of pathogens has become an essential challenge in the biomedical field. Herein a highly selective platform based on aptamer-gated nano materials for specific Staphylococcus aureus detection is presented. In the proposed design, a nanoporous anodic alumina (NAA) scaffold is loaded with the fluorescent indicator rhodamine B, while pores entrances are capped by a DNA aptamer which selectively recognizes S. aureus cells in less than 1 h. When S. aureus cells are present, the solid is selectively uncapped, and the dye is released to the medium. This nanodevice allows the detection of bacterial concentrations between 2 and 5 CFU mL-1 (in buffer and blood, respectively) and it has demonstrated excellent behavior in terms of specificity and robustness. A set of 25 different clinical samples are analyzed using this simple procedure obtaining excellent results, which agree with conventional hospital reference techniques for the identification of S. aureus. This new method is sensitive, rapid and low cost, and avoids steps such as polymerase chain amplification reaction, which makes it suitable for use in point-of-care detection systems.
- Published
- 2020
16. Triplex hybridization-based nanosystem for the rapid screening of pneumocystis pneumonia in clinical samples
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Agencia Estatal de Investigación, European Regional Development Fund, Ministerio de Ciencia, Innovación y Universidades, Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Pla, Luis, Aviñó, Anna, Eritja, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Vicente, Calderón, Enrique J., Aznar, Elena, Martínez-Máñez, Ramón, Santiago Felipe, Sara, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Agencia Estatal de Investigación, European Regional Development Fund, Ministerio de Ciencia, Innovación y Universidades, Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Pla, Luis, Aviñó, Anna, Eritja, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Vicente, Calderón, Enrique J., Aznar, Elena, Martínez-Máñez, Ramón, and Santiago Felipe, Sara
- Abstract
[EN] Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.
- Published
- 2020
17. Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples
- Author
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Universidad de Sevilla. Departamento de Medicina, Universidad de Sevilla. CTS-458: Investigación Clínica y Básica en Medicina Interna, Calidad y Continuidad Asistencial, Pla, Luis, Aviñó, Anna, Eritja, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Vicente, Calderón Sandubete, Enrique José, Santiago-Felipe, Sara, Universidad de Sevilla. Departamento de Medicina, Universidad de Sevilla. CTS-458: Investigación Clínica y Básica en Medicina Interna, Calidad y Continuidad Asistencial, Pla, Luis, Aviñó, Anna, Eritja, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Vicente, Calderón Sandubete, Enrique José, and Santiago-Felipe, Sara
- Abstract
Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.
- Published
- 2020
18. Triplex Hybridization-Based Nanosystem for the Rapid Screening of Pneumocystis Pneumonia in Clinical Samples
- Author
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Eritja Casadellà, Ramón [0000-0001-5383-9334], Pla, Luis, Aviñó, Anna, Eritja Casadellà, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Javier, Calderón, Enrique J., Aznar, Elena, Martínez-Máñez, Ramón, Santiago-Felipe, Sara, Eritja Casadellà, Ramón [0000-0001-5383-9334], Pla, Luis, Aviñó, Anna, Eritja Casadellà, Ramón, Ruiz-Gaitán, Alba, Pemán, Javier, Friaza, Javier, Calderón, Enrique J., Aznar, Elena, Martínez-Máñez, Ramón, and Santiago-Felipe, Sara
- Abstract
Pneumocystis pneumonia (PcP) is a disease produced by the opportunistic infection of the fungus Pneumocystis jirovecii. As delayed or unsuitable treatments increase the risk of mortality, the development of rapid and accurate diagnostic tools for PcP are of great importance. Unfortunately, current standard methods present severe limitations and are far from adequate. In this work, a time-competitive, sensitive and selective biosensor based on DNA-gated nanomaterials for the identification of P. jirovecii is presented. The biosensor consists of a nanoporous anodic alumina (NAA) scaffold which pores are filled with a dye reporter and capped with specific DNA oligonucleotides. In the presence of P. jirovecii genomic DNA, the gated biosensor is open, and the cargo is delivered to the solution where it is monitored through fluorescence spectroscopy. The use of capping oligonucleotides able to form duplex or triplex with P. jirovecii DNA is studied. The final diagnostic tool shows a limit of detection (LOD) of 1 nM of target complementary DNA and does not require previous amplification steps. The method was applied to identify DNA from P. jirovecii in unmodified bronchoalveolar lavage, nasopharyngeal aspirates, and sputum samples in 60 min. This is a promising alternative method for the routinely diagnosis of Pneumocystis pneumonia.
- Published
- 2020
19. Selective and Sensitive Probe Based in Oligonucleotide-Capped Nanoporous Alumina for the Rapid Screening of Infection Produced byCandida albicans
- Author
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Ribes, Àngela, primary, Aznar, Elena, additional, Santiago-Felipe, Sara, additional, Xifre-Perez, Elisabet, additional, Tormo-Mas, María Ángeles, additional, Pemán, Javier, additional, Marsal, Lluis F., additional, and Martínez-Máñez, Ramón, additional
- Published
- 2019
- Full Text
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20. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis
- Author
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Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, and Maquieira Catala, Ángel
- Subjects
DNA, Bacterial ,Streptavidin ,DNA, Plant ,Loop-mediated isothermal amplification ,Recombinase Polymerase Amplification ,Enzyme-Linked Immunosorbent Assay ,DNA-Directed DNA Polymerase ,Biochemistry ,Food safety ,Analytical Chemistry ,law.invention ,Recombinases ,Isothermal amplification ,chemistry.chemical_compound ,Fusarium ,Salmonella ,law ,QUIMICA ANALITICA ,Recombinase ,Environmental Chemistry ,Cronobacter ,DNA, Fungal ,Spectroscopy ,Polymerase chain reaction ,Polymerase ,Chromatography ,biology ,GMO ,Pathogen ,Allergen ,Temperature ,DNA ,Allergens ,Plants ,Plants, Genetically Modified ,biology.organism_classification ,Molecular biology ,chemistry ,Biotinylation ,Food Microbiology ,biology.protein ,ELISA ,Nucleic Acid Amplification Techniques ,Food Analysis - Abstract
[EN] Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40 degrees C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. (C) 2013 Elsevier B.V. All rights reserved., This research has been funded through Projects GV/2009/028 (Generalitat Valenciana) and CTQ/2010/15943 (MICINN). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies. The fungal cultures were supplied by B. Mora-Sala and J. Garcia-Jimenez from the Instituto Agroforestal Mediterraneo, UPV.
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- 2014
- Full Text
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21. Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Universitat Politècnica de València. Departamento de Ingeniería Mecánica y de Materiales - Departament d'Enginyeria Mecànica i de Materials, Generalitat Valenciana, Generalitat de Catalunya, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Ministerio de Educación, Cultura y Deporte, European Social Fund, European Regional Development Fund, Ribes, Àngela, Santiago Felipe, Sara, Aviñó, Anna, Candela-Noguera, Vicente, Eritja, Ramón, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, Aznar, Elena, Universitat Politècnica de València. Departamento de Química - Departament de Química, Universitat Politècnica de València. Departamento de Ingeniería Mecánica y de Materiales - Departament d'Enginyeria Mecànica i de Materials, Generalitat Valenciana, Generalitat de Catalunya, Instituto de Salud Carlos III, Ministerio de Ciencia e Innovación, Ministerio de Economía y Competitividad, Ministerio de Educación, Cultura y Deporte, European Social Fund, European Regional Development Fund, Ribes, Àngela, Santiago Felipe, Sara, Aviñó, Anna, Candela-Noguera, Vicente, Eritja, Ramón, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, and Aznar, Elena
- Abstract
[EN] The development of new strategies to detect microRNAs (miRNAS) has become an important challenge in thebiomedical ¿eld. We report herein the use of oligonucleotide-gated silica nanoparticles for the detection ofmiRNA-145. In the proposed design, mesoporous silica nanoparticles (MSNs) are loaded with a ¿uorescentreporter (rhodamine B) and pores are blocked by speci¿c DNA oligonucleotides. The opening of the gated systemand dye delivery is selectively controlled by DNA-miRNA recognition. Moreover, the use of DNA capture probesable to form duplex or triplex structures between target miRNA and the complementary oligonucleotides hasbeen studied. By this simple procedure a limit of detection as low as 0.25 pM was found for miRNA. The methodwas successfully applied to detect miRNA-145 in serum samples, which demonstrates the high potential of thesecapped materials to detect miRNA for diagnostic purposes
- Published
- 2018
22. Design of oligonucleotide-capped mesoporous silica nanoparticles for the detection of miRNA-145 by duplex and triplex formation
- Author
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Ministerio de Economía y Competitividad (España), Ribes, Àngela, Santiago-Felipe, Sara, Aviñó, Anna, Candela-Noguera, Vicente, Eritja Casadellà, Ramón, Sancenón, Félix, Martínez-Máñez, Ramón, Aznar, Elena, Ministerio de Economía y Competitividad (España), Ribes, Àngela, Santiago-Felipe, Sara, Aviñó, Anna, Candela-Noguera, Vicente, Eritja Casadellà, Ramón, Sancenón, Félix, Martínez-Máñez, Ramón, and Aznar, Elena
- Abstract
The development of new strategies to detect microRNAs (miRNAS) has become an important challenge in the biomedical field. We report herein the use of oligonucleotide-gated silica nanoparticles for the detection of miRNA-145. In the proposed design, mesoporous silica nanoparticles (MSNs) are loaded with a fluorescent reporter (rhodamine B) and pores are blocked by specific DNA oligonucleotides. The opening of the gated system and dye delivery is selectively controlled by DNA-miRNA recognition. Moreover, the use of DNA capture probes able to form duplex or triplex structures between target miRNA and the complementary oligonucleotides has been studied. By this simple procedure a limit of detection as low as 0.25 pM was found for miRNA. The method was successfully applied to detect miRNA-145 in serum samples, which demonstrates the high potential of these capped materials to detect miRNA for diagnostic purposes.
- Published
- 2018
23. Comparison of DNA amplification methods for simultaneous detection of Salmonella spp. and Cronobacter sakazakii in milk
- Author
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Santiago Felipe, Sara
- Subjects
Multiple displacement amplification (MDA) ,Recombinase Polymerase Amplification (RPA) ,Salmonella spp ,QUIMICA ANALITICA ,Máster Universitario en Sensores para Aplicaciones Industriales-Màster Universitari en Sensors per a Aplicacions Industrials ,DNA amplification methods ,Cronobacter Sakazakii - Abstract
The control and detection of foodborne pathogens is a major problem in public health. Therefore, the development of rapid and economical diagnostic method along the food chain has become a priority. Among the available methodologies, those based on the detection of nucleic acids provide certain advantages such as high sensitivity speed, selectivity, and the ability to simultaneously detect multiple microorganisms. However, the amount of pathogen DNA present in a food is minimal, so these methods must incorporate an amplification stage that achieves the proper amount for their detection. The polymerase chain reaction (PCR) is the common technique, although this method has the requirement of thermocycling. In this master project two enzymatic reactions that enable the amplification of DNA under isothermal conditions have been studied. The work focuses on the comparison of conventional PCR with two isothermal amplification methods ‐recombinase polymerase (RPA) and multiple displacement amplification (MDA)‐. These methods, applied to the detection of Salmonella spp. and Cronobacter sakazakii, have shown excellent amplification yields of the target sequences. In addition, the inhibitory effect of some matrix components on the enzymes has been studied. The simultaneous detection of two pathogens has been made in milk samples, analyzing the amplified products by hybridization assays with oligonucleotide probes immobilized on the surface of a DVD in microarray format. The results have been satisfactory in terms of reproducibility and sensitivity. It has been shown that the use of other alternative enzymes and low cost heating equipments allow DNA amplification at constant temperature. Moreover, the combination with compact disc technology increases the advantages, demonstrating the validity of the proposed biosensor in routine applications in the area of food safety.
- Published
- 2016
24. Two New Fluorogenic Aptasensors Based on Capped Mesoporous Silica Nanoparticles to Detect Ochratoxin A
- Author
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Ribes, Àngela, primary, Santiago-Felipe, Sara, additional, Bernardos, Andrea, additional, Marcos, M. Dolores, additional, Pardo, Teresa, additional, Sancenón, Félix, additional, Martínez-Máñez, Ramón, additional, and Aznar, Elena, additional
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- 2017
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25. Selective and Sensitive Probe Based in Oligonucleotid e-Capped Nanoporous Alumina for the Rapid Screening of Infection Produced by Candida albicans.
- Author
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Ribes, Àngela, Aznar, Elena, Santiago-Felipe, Sara, Xifre-Perez, Elisabet, Tormo-Mas, María Ángeles, Pemán, Javier, Marsal, Lluis F., and Martínez-Máñez, Ramón
- Published
- 2019
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26. Two New Fluorogenic Aptasensors Based on Capped Mesoporous Silica Nanoparticles to Detect Ochratoxin A
- Author
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Universitat Politècnica de València. Instituto de Reconocimiento Molecular y Desarrollo Tecnológico - Institut de Reconeixement Molecular i Desenvolupament Tecnològic, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Economía, Industria y Competitividad, Ribes, Àngela, Santiago Felipe, Sara, Bernardos Bau, Andrea, Marcos Martínez, María Dolores, Pardo Vicente, María Teresa, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, Aznar, Elena, Universitat Politècnica de València. Instituto de Reconocimiento Molecular y Desarrollo Tecnológico - Institut de Reconeixement Molecular i Desenvolupament Tecnològic, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Economía, Industria y Competitividad, Ribes, Àngela, Santiago Felipe, Sara, Bernardos Bau, Andrea, Marcos Martínez, María Dolores, Pardo Vicente, María Teresa, Sancenón Galarza, Félix, Martínez-Máñez, Ramón, and Aznar, Elena
- Abstract
[EN] Aptamers have been used as recognition elements for several molecules due to their great affinity and selectivity. Additionally, mesoporous nanomaterials have demonstrated great potential in sensing applications. Based on these concepts, we report herein the use of two aptamer¿capped mesoporous silica materials for the selective detection of ochratoxin¿A (OTA). A specific aptamer for OTA was used to block the pores of rhodamine¿B¿loaded mesoporous silica nanoparticles. Two solids were prepared in which the aptamer capped the porous scaffolds by using a covalent or electrostatic approach. Whereas the prepared materials remained capped in water, dye delivery was selectively observed in the presence of OTA. The protocol showed excellent analytical performance in terms of sensitivity (limit of detection: 0.5¿0.05¿nm), reproducibility, and selectivity. Moreover, the aptasensors were tested for OTA detection in commercial foodstuff matrices, which demonstrated their potential applicability in real samples
- Published
- 2017
27. Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Educación, Generalitat Valenciana, Ministerio de Economía y Competitividad, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Carrascosa Rubio, Javier, Puchades, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Educación, Generalitat Valenciana, Ministerio de Economía y Competitividad, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Carrascosa Rubio, Javier, Puchades, Rosa, and Maquieira Catala, Ángel
- Abstract
An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2mm diameter and 1.1mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proof-of-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15min, with high selectivity and sensitivity (5CFU/mL and 10µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses.
- Published
- 2016
28. Comparison of DNA amplification methods for simultaneous detection of Salmonella spp. and Cronobacter sakazakii in milk
- Author
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Tortajada Genaro, Luis Antonio, Puchades Pla, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería del Diseño - Escola Tècnica Superior d'Enginyeria del Disseny, Santiago Felipe, Sara, Tortajada Genaro, Luis Antonio, Puchades Pla, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería del Diseño - Escola Tècnica Superior d'Enginyeria del Disseny, and Santiago Felipe, Sara
- Abstract
The control and detection of foodborne pathogens is a major problem in public health. Therefore, the development of rapid and economical diagnostic method along the food chain has become a priority. Among the available methodologies, those based on the detection of nucleic acids provide certain advantages such as high sensitivity speed, selectivity, and the ability to simultaneously detect multiple microorganisms. However, the amount of pathogen DNA present in a food is minimal, so these methods must incorporate an amplification stage that achieves the proper amount for their detection. The polymerase chain reaction (PCR) is the common technique, although this method has the requirement of thermocycling. In this master project two enzymatic reactions that enable the amplification of DNA under isothermal conditions have been studied. The work focuses on the comparison of conventional PCR with two isothermal amplification methods ‐recombinase polymerase (RPA) and multiple displacement amplification (MDA)‐. These methods, applied to the detection of Salmonella spp. and Cronobacter sakazakii, have shown excellent amplification yields of the target sequences. In addition, the inhibitory effect of some matrix components on the enzymes has been studied. The simultaneous detection of two pathogens has been made in milk samples, analyzing the amplified products by hybridization assays with oligonucleotide probes immobilized on the surface of a DVD in microarray format. The results have been satisfactory in terms of reproducibility and sensitivity. It has been shown that the use of other alternative enzymes and low cost heating equipments allow DNA amplification at constant temperature. Moreover, the combination with compact disc technology increases the advantages, demonstrating the validity of the proposed biosensor in routine applications in the area of food safety.
- Published
- 2016
29. Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Economía y Competitividad, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Economía y Competitividad, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, and Maquieira Catala, Ángel
- Abstract
An integrated method for the parallelized detection of multiple DNA target sequences is presented by using microstructures in a digital versatile disc (DVD). Samples and reagents were managed by using both the capillary and centrifugal forces induced by disc rotation. Recombinase polymerase amplification (RPA), in a bridge solid phase format, took place in separate wells, which thereby modified their optical properties. Then the DVD drive reader recorded the modifications of the transmitted laser beam. The strategy allowed tens of genetic determinations to be made simultaneously within < 2 h, with small sample volumes (3 mu L), low manipulation and at low cost. The method was applied to high-throughput screening of relevant safety threats (allergens, GMOs and pathogenic bacteria) in food samples. Satisfactory results were obtained in terms of sensitivity (48.7 fg of DNA) and reproducibility (below 18 %). This scheme warrants cost-effective multiplex amplification and detection and is perceived to represent a viable tool for screening of nucleic acid targets.
- Published
- 2016
30. One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method
- Author
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Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Morais, Sergi, Puchades, Rosa, and Maquieira Catala, Ángel
- Subjects
In situ ,Materials science ,Fold (higher-order function) ,Pathogen ,Metals and Alloys ,Analytical chemistry ,Compact disc ,Recombinase Polymerase Amplification ,Microarray ,Condensed Matter Physics ,Dna amplification ,Optical scanning ,Isothermal process ,Surfaces, Coatings and Films ,Electronic, Optical and Magnetic Materials ,QUIMICA ANALITICA ,Materials Chemistry ,Isothermal solid-phase amplification ,Multiplex ,Electrical and Electronic Engineering ,Instrumentation - Abstract
[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved., This research has been funded through Projects GVA-PROMETEO/2010/008 (Generalitat Valenciana) and CTQ/2013/ 45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for her PhD studies.
- Published
- 2014
31. Comparison of DNA amplification methods for simultaneous detection of Salmonella spp. and Cronobacter sakazakii in milk
- Author
-
Santiago Felipe, Sara|||0000-0002-1731-815X
- Subjects
Multiple displacement amplification (MDA) ,Recombinase Polymerase Amplification (RPA) ,Salmonella spp ,QUIMICA ANALITICA ,Máster Universitario en Sensores para Aplicaciones Industriales-Màster Universitari en Sensors per a Aplicacions Industrials ,DNA amplification methods ,Cronobacter Sakazakii - Abstract
The control and detection of foodborne pathogens is a major problem in public health. Therefore, the development of rapid and economical diagnostic method along the food chain has become a priority. Among the available methodologies, those based on the detection of nucleic acids provide certain advantages such as high sensitivity speed, selectivity, and the ability to simultaneously detect multiple microorganisms. However, the amount of pathogen DNA present in a food is minimal, so these methods must incorporate an amplification stage that achieves the proper amount for their detection. The polymerase chain reaction (PCR) is the common technique, although this method has the requirement of thermocycling. In this master project two enzymatic reactions that enable the amplification of DNA under isothermal conditions have been studied. The work focuses on the comparison of conventional PCR with two isothermal amplification methods ‐recombinase polymerase (RPA) and multiple displacement amplification (MDA)‐. These methods, applied to the detection of Salmonella spp. and Cronobacter sakazakii, have shown excellent amplification yields of the target sequences. In addition, the inhibitory effect of some matrix components on the enzymes has been studied. The simultaneous detection of two pathogens has been made in milk samples, analyzing the amplified products by hybridization assays with oligonucleotide probes immobilized on the surface of a DVD in microarray format. The results have been satisfactory in terms of reproducibility and sensitivity. It has been shown that the use of other alternative enzymes and low cost heating equipments allow DNA amplification at constant temperature. Moreover, the combination with compact disc technology increases the advantages, demonstrating the validity of the proposed biosensor in routine applications in the area of food safety.
- Published
- 2012
32. Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs)
- Author
-
Tortajada-Genaro, Luis A., Santiago-Felipe, Sara, Amasia, Mary, Russom, Aman, Maquieira, Angel, Tortajada-Genaro, Luis A., Santiago-Felipe, Sara, Amasia, Mary, Russom, Aman, and Maquieira, Angel
- Abstract
A new advancement in massive DNA-based screening in limited-resource settings is demonstrated through the incorporation of easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform isothermal recombinase polymerase amplification (RPA) in a microarray format. Standard un-modified DVD discs and commercial drives are used for the low-cost detection method. DNA primers were printed in a microarray format on the polycarbonate surfaces of DVDs with integrated control spots to guarantee the absence of false-negatives and false-positives. The solid-phase amplification assay, including the washing protocols and development reaction, was performed by the dispensation of solutions through the inlet and by controlling the flow-movement by DVD drive centrifugation. The final disc with reaction products was inserted into a DVD player and microarray images were captured and automatically processed. This simple approach was applied for the screening of genetically modified organisms (GMOs) in food samples. The limit of detection was 7 mu g g(-1), which is well below the EU regulation limit for GMOs in food products. Therefore, the only required materials for food safety monitoring were standard store-bought DVDs, plastic chambers, tips, pipettes, an oven, and a standard DVD drive. The proposed strategy allows an integrated microarray system with low manipulation, reduced sample volume, and portability, which are beneficial for low-resource settings., QC 20150807
- Published
- 2015
- Full Text
- View/download PDF
33. Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs)
- Author
-
Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural - Escola Tècnica Superior d'Enginyeria Agronòmica i del Medi Natural, Universitat Politècnica de València. Departamento de Química - Departament de Química, European Commission, Universitat Politècnica de València, Generalitat Valenciana, Ministerio de Economía y Competitividad, Tortajada-Genaro, Luis Antonio, Santiago Felipe, Sara, Amasia, Mary, Russom, Aman, Maquieira Catala, Ángel, Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería Agronómica y del Medio Natural - Escola Tècnica Superior d'Enginyeria Agronòmica i del Medi Natural, Universitat Politècnica de València. Departamento de Química - Departament de Química, European Commission, Universitat Politècnica de València, Generalitat Valenciana, Ministerio de Economía y Competitividad, Tortajada-Genaro, Luis Antonio, Santiago Felipe, Sara, Amasia, Mary, Russom, Aman, and Maquieira Catala, Ángel
- Abstract
[EN] A new advancement in massive DNA-based screening in limited-resource settings is demonstrated through the incorporation of easy-to-fabricate microfluidic chambers on digital versatile discs (DVDs) to perform isothermal recombinase polymerase amplification (RPA) in a microarray format. Standard un-modified DVD discs and commercial drives are used for the low-cost detection method. DNA primers were printed in a microarray format on the polycarbonate surfaces of DVDs with integrated control spots to guarantee the absence of false-negatives and false-positives. The solid-phase amplification assay, including the washing protocols and development reaction, was performed by the dispensation of solutions through the inlet and by controlling the flow-movement by DVD drive centrifugation. The final disc with reaction products was inserted into a DVD player and microarray images were captured and automatically processed. This simple approach was applied for the screening of genetically modified organisms (GMOs) in food samples. The limit of detection was 7 mu g g(-1), which is well below the EU regulation limit for GMOs in food products. Therefore, the only required materials for food safety monitoring were standard store-bought DVDs, plastic chambers, tips, pipettes, an oven, and a standard DVD drive. The proposed strategy allows an integrated microarray system with low manipulation, reduced sample volume, and portability, which are beneficial for low-resource settings.
- Published
- 2015
34. Isothermal DNA amplification strategies for duplex microorganism detection
- Author
-
Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Economía y Competitividad, Generalitat Valenciana, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Morais, Sergi, Puchades, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Economía y Competitividad, Generalitat Valenciana, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Morais, Sergi, Puchades, Rosa, and Maquieira Catala, Ángel
- Abstract
[EN] A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2 8.6 108 fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (101 102 CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.
- Published
- 2015
35. Integración de técnicas basadas en ADN para el desarrollo de biosensores aplicados en seguridad alimentaria
- Author
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Maquieira Catala, Ángel, Puchades Pla, Rosa, Tortajada Genaro, Luis Antonio, Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería del Diseño - Escola Tècnica Superior d'Enginyeria del Disseny, Santiago Felipe, Sara, Maquieira Catala, Ángel, Puchades Pla, Rosa, Tortajada Genaro, Luis Antonio, Universitat Politècnica de València. Escuela Técnica Superior de Ingeniería del Diseño - Escola Tècnica Superior d'Enginyeria del Disseny, and Santiago Felipe, Sara
- Abstract
Tesis por compendio, [EN] Food security is guaranteed when there is sufficient, safe and nutritious food. This assurance must be satisfied throughout the entire production process, which is known as "safety from farm to fork". This results in a new way of addressing the problem with a global and comprehensive approach. To address this challenge, molecular techniques based on the use of nucleic acids are used in the analysis of certain food threatens, such as allergens, microorganisms, genetically modified organisms (GMOs), or food authentication. However, some of the described methods still have limitations, since they are expensive, complicated, and require specialized staff and equipment. Alternatively, biosensor technology provides reliable results in a simpler and faster way and with and added capabilities such as portability and automation, allowing to perform the analysis directly at points-of-control (POC). This thesis has focused on developing a biosensor system, based on compact disc technology, for the detection of nucleic acids in food safety applications and adaptable to POC needs. The carried out investigations have yielded new insights into gene technology, making interesting methodological contributions characterized by miniaturization, integration and automation. The first part of the research deals with the simplification of the amplification step, eluding the thermocycling by using alternatives to the polymerase chain reaction (PCR). To this end, two isothermal amplification techniques have been studied: the recombinase polymerase amplification (RPA) and the multiple displacement amplification (MDA). The detection was performed by hybridization assays with DNA probes immobilized in microarray format on the polycarbonate surface of a DVD. Furthermore, RPA amplification has been combined with detection by an immunoenzymatic assay (ELISA) for the simultaneous detection of multiple analytes. In another approach, amplification and hybridization have been integrated in a sin, [ES] La seguridad alimentaria está garantizada cuando se dispone de alimentos suficientes, nutritivos e inocuos. Esta garantía, además, ha de cumplirse a lo largo de todo el proceso productivo, lo que se conoce como "seguridad de la granja a la mesa". Nace así una nueva forma de abordar el problema, con un enfoque global y un tratamiento integral. Para afrontar este reto, las técnicas moleculares basadas en el empleo de ácidos nucleicos son, actualmente, utilizadas en la detección de amenazas alimentarias, como por ejemplo, alérgenos, microorganismos, organismos genéticamente modificados (OGM), o la autentificación de especies. Sin embargo, muchos de los métodos en uso presentan limitaciones, ya que son costosos, complicados, y requieren personal y equipamiento especializado. La tecnología de biosensores es una aproximación adecuada, ya que proporciona resultados fiables de manera sencilla y rápida, y con capacidades añadidas como portabilidad y automatización para llevar a cabo los ensayos directamente en puntos de control (POC). Esta tesis se ha centrado en el desarrollo de un sistema biosensor, basado en la tecnología de disco compacto, para la detección de ácidos nucleicos en aplicaciones de seguridad alimentaria adaptable a POC. Las investigaciones llevadas a cabo han permitido obtener nuevos conocimientos en tecnologías génicas, pudiendo efectuar aportaciones metodológicas de interés caracterizadas por su miniaturización, integración y automatización. En este sentido, una parte de la investigación aborda la simplificación de la etapa de amplificación del ADN diana, eludiendo el termociclado mediante el empleo de técnicas alternativas a la reacción en cadena de la polimerasa (PCR). Para ello, se han estudiado dos técnicas de amplificación isoterma, la amplificación por recombinasa polimerasa (RPA) y la amplificación por desplazamiento múltiple (MDA). La detección se lleva a cabo mediante ensayos de hibridación con sondas de ADN inmovilizadas en formato micromat, [CA] La seguretat alimentària està garantida quan es disposa d'aliments suficients, nutritius i innocus. Esta garantia, a més, ha de complir-se al llarg de tot el procés productiu, el que es coneix com a "seguretat de la granja a la taula". Naix així una nova forma d'abordar el problema, amb un enfocament global i un tractament integral. Per a afrontar este repte, les tècniques moleculars basades en la utilització d'àcids nucleics són, actualment, usades en l'anàlisi de certes amenaces alimentàries, com per exemple, la detecció d'al·lèrgens, microorganismes, organismes genèticament modificats (OGM), o l'autentificació de determinades espècies. No obstant això, molts dels mètodes en ús presenten limitacions, ja que són costosos, complicats, i requerixen personal i equipament especialitzat. La tecnologia de biosensors és una aproximació adequada, ja que proporciona resultats fiables de manera senzilla i ràpida, i amb capacitats afegides com portabilitat i automatització per a dur a terme els assajos directament en punts de control (POC). Esta tesi s'ha centrat en el desenvolupament d'un sistema biosensor, basat en la tecnologia de disc compacte, per a la detecció d'àcids nucleics en aplicacions de seguretat alimentària adaptable a POC. Les investigacions dutes a terme han permés obtindre nous coneixements en tecnologies gèniques, podent efectuar aportacions metodològiques d'interés caracteritzades per la seua miniaturització, integració i automatització. En este sentit, una part de la investigació aborda la simplificació de l'etapa d'amplificació de l'ADN diana, eludint el termociclat per mitjà de l'utilització de tècniques alternatives a la reacció en cadena de la polimerasa (PCR). Per a això, s'han estudiat dos tècniques d'amplificació isoterma, l'amplificació per recombinasa polimerasa (RPA) i l'amplificació per desplaçament múltiple (MDA). La detecció es du a terme per mitjà d'assajos d'hibridació amb sondes d'ADN immobilitzades en format micromatriu sobre la supe
- Published
- 2015
36. Isothermal DNA amplification strategies for duplex microorganism detection
- Author
-
Santiago-Felipe, Sara, primary, Tortajada-Genaro, Luis Antonio, additional, Morais, Sergi, additional, Puchades, Rosa, additional, and Maquieira, Ángel, additional
- Published
- 2015
- Full Text
- View/download PDF
37. Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs)
- Author
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Tortajada-Genaro, Luis A., primary, Santiago-Felipe, Sara, additional, Amasia, Mary, additional, Russom, Aman, additional, and Maquieira, Ángel, additional
- Published
- 2015
- Full Text
- View/download PDF
38. Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Ciencia e Innovación, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Ciencia e Innovación, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Puchades, Rosa, and Maquieira Catala, Ángel
- Abstract
[EN] Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40 degrees C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings. (C) 2013 Elsevier B.V. All rights reserved.
- Published
- 2014
39. One-pot isothermal DNA amplification Hybridisation and detection by a disc-based method
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Economía y Competitividad, Generalitat Valenciana, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Morais, Sergi, Puchades, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Ministerio de Economía y Competitividad, Generalitat Valenciana, Ministerio de Educación y Ciencia, Santiago Felipe, Sara, Tortajada-Genaro, Luis Antonio, Morais, Sergi, Puchades, Rosa, and Maquieira Catala, Ángel
- Abstract
[EN] An integrated sensor comprising isothermal DNA amplification and in situ detection is presented. The method principle is based on recombinase polymerase amplification (RPA) and detection in the microarray format by compact disc technology as a high-throughput sensing platform. Primers were immobilised on the polycarbonate surface of digital versatile discs (DVD) and, after hemi-nested amplification, multiplexing identification of each tethered product was achieved by optical scanning with a 650 nm-laser of the DVD drive. The efficiency of one-pot hybridisation/elongation/detection depended strongly on probedensity and other factors such as the concentration of the unbound primers present in solution. The optimised conditions provided equivalent amplification factors (7.3 x 10(8) -8.9 x 10(8) fold) to those obtained by conventional reactions performed in vials. The proposed method was applied to Salmonella detection (generic by hns and oriC genes, and specific for subspecies I by STM4507 gene). A triplex assay was satisfactorily compared to the non-integrated protocols. Food and vaccine samples were analysed in a shorter time with less handling. The results indicate that the multiplex DVD assay is a simple, competitive, isothermal, portable system that is particularly useful for microbiological routine analysis. (C) 2014 Elsevier B.V. All rights reserved.
- Published
- 2014
40. Multiplex DNA Detection of Food Allergens on a Digital Versatile Disk
- Author
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Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Ciencia e Innovación, Ministerio de Educación y Ciencia, Tortajada Genaro, Luis Antonio, Santiago Felipe, Sara, Morais Ezquerro, Sergi Beñat, GABALDÓN, JOSE ANTONIO, Puchades Pla, Rosa, Maquieira Catala, Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química, Generalitat Valenciana, Ministerio de Ciencia e Innovación, Ministerio de Educación y Ciencia, Tortajada Genaro, Luis Antonio, Santiago Felipe, Sara, Morais Ezquerro, Sergi Beñat, GABALDÓN, JOSE ANTONIO, Puchades Pla, Rosa, and Maquieira Catala, Ángel
- Abstract
The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut (Corylus avellana L.), peanut (Arachis hypogaea), and soybean (Glycine max) in foods. After DNA extraction, multiplex PCR was set up using S'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with S'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 mu g/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.
- Published
- 2012
41. Aplicación de la Ingeniería Emocional para potenciar las sensaciones en la gastronomía
- Author
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Santiago Felipe, Sara, Marzo Roselló, Raquel, Ferrís Oñate, Javier, Soriano García, Carolina, Such Pérez, Mª José, Sánchez Lacuesta, Javier, Santiago Felipe, Sara, Marzo Roselló, Raquel, Ferrís Oñate, Javier, Soriano García, Carolina, Such Pérez, Mª José, and Sánchez Lacuesta, Javier
- Abstract
Instituto de Biomecánica de Valencia (IBV) and Masía Xamandreu have applied in a pioneer way Emotional Engineering techniques in the gastronomy sector, which is one of the most important in Comunidad Valenciana. These techniques enable to study the relationship between the emotional concepts that the Company wants to transmit and the messages perceived by the clients through the visual assessment of food arrangements. IBV uses Emotional Engineering methodologies to obtain knowledge that allow relating technical, functional and aesthetic characteristics of the products with user's emotions. The application of this methodology in food arrangements is very innovative internationally, and allows to understand the relationship between the brand values of Masía Xamandreu and plate design. Thus it's possible to determine which combination of design elements maximizes this perception, and therefore increases the probability that consumers choose the restaurant. Masía Xamandreu's web page (www.masiaxamandreu.com) incorporated the results of this project, as it is a very important tool in the purchase experience, especially in early stages like search and selection of restaurants, where brand values play an important role in consumer choice., El Instituto de Biomecánica de Valencia (IBV) y la Masía Xamandreu han aplicado la ingeniería emocional para estudiar la relación entre los conceptos emocionales que esta empresa pretende transmitir y los mensajes percibidos por los clientes a través de la evaluación visual del emplatado. Éste es un estudio pionero en el sector gastronómico, de enorme importancia en la Comunidad Valenciana y en España. La Ingeniería Emocional permite relacionar las características técnicas, funcionales y estéticas de los productos con las emociones que despiertan en los usuarios. La aplicación de esta metodología en el ámbito de la restauración es absolutamente novedosa a nivel internacional, y nos permite conocer qué combinación de elementos de emplatado aumenta la probabilidad de elección por parte del consumidor, en coherencia con los valores de marca de la empresa. Los resultados han sido incorporados en la página web de la Masía Xamandreu (www.masiaxamandreu.com) para estimular al cliente potencial en la fase de búsqueda y selección de restaurante para sus celebraciones.
- Published
- 2009
42. Multiplex DNA Detection of Food Allergens on a Digital Versatile Disk
- Author
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Tortajada-Genaro, Luis A., primary, Santiago-Felipe, Sara, additional, Morais, Sergi, additional, Gabaldón, José Antonio, additional, Puchades, Rosa, additional, and Maquieira, Ángel, additional
- Published
- 2011
- Full Text
- View/download PDF
43. Integración de técnicas basadas en ADN para el desarrollo de biosensores aplicados en seguridad alimentaria
- Author
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Santiago Felipe, Sara, primary
- Full Text
- View/download PDF
44. Selective and Sensitive Probe Based in Oligonucleotide-Capped Nanoporous Alumina for the Rapid Screening of Infection Produced by Candida albicans.
- Author
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Ribes À, Aznar E, Santiago-Felipe S, Xifre-Perez E, Tormo-Mas MÁ, Pemán J, Marsal LF, and Martínez-Máñez R
- Subjects
- Candida albicans genetics, Candida albicans physiology, DNA, Fungal analysis, DNA, Fungal chemistry, Humans, Limit of Detection, Time Factors, Aluminum Oxide chemistry, Biosensing Techniques methods, Candida albicans isolation & purification, Nanopores, Oligonucleotides chemistry
- Abstract
A robust, sensitive, and time-competitive system to detect Candida albicans in less than 30 min in clinical samples based in capped nanoporous anodic alumina (NAA) is developed. In the proposed design, NAA pores are loaded with rhodamine B and then blocked with an oligonucleotide that is able to recognize C. albicans DNA. The capped material shows negligible cargo release, whereas dye delivery is selectively accomplished when genomic DNA from C. albicans is present. This procedure has been successfully applied to detect C. albicans in clinical samples from patients infected with this yeast. When compared with classical C. albicans detection methods, the proposed probe has a short assay time, high sensitivity and selectivity, demonstrating the high potential of this simple design for the diagnosis of infection produced by C. albicans.
- Published
- 2019
- Full Text
- View/download PDF
45. Multiplex DNA detection of food allergens on a digital versatile disk.
- Author
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Tortajada-Genaro LA, Santiago-Felipe S, Morais S, Gabaldón JA, Puchades R, and Maquieira Á
- Subjects
- Allergens analysis, Antigens, Plant analysis, Arachis chemistry, Corylus chemistry, Food Hypersensitivity prevention & control, Humans, Multiplex Polymerase Chain Reaction instrumentation, Glycine max chemistry, Allergens genetics, Antigens, Plant genetics, Arachis genetics, Corylus genetics, Multiplex Polymerase Chain Reaction methods, Glycine max genetics
- Abstract
The development of a DNA microarray method on a digital versatile disk (DVD) is described for the simultaneous detection of traces of hazelnut ( Corylus avellana L.), peanut ( Arachis hypogaea ), and soybean ( Glycine max ) in foods. After DNA extraction, multiplex PCR was set up using 5'-labeled specific primers for Cor a 1, Ar h 2, and Le genes, respectively. Digoxin-labeled PCR products were detected by hybridization with 5'-biotinylated probes immobilized on a streptavidin-modified DVD surface. The reaction product attenuates the signal intensity of the laser that reached the DVD drive used as detector, correlating well with the amount of amplified sequence. Analytical performances showed a detection limit of 1 μg/g and good assay reproducibility (RSD 8%), suitable for the simultaneous detection of the three targeted allergens. The developed methodology was tested with several commercially available foodstuffs, demonstrating its applicability. The results were in good agreement, in terms of sensitivity and reproducibility, with those obtained with ELISA, PCR-gel agarose electrophoresis, and RT-PCR.
- Published
- 2012
- Full Text
- View/download PDF
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