Implementación de plataformas biosensoras ópticas basadas en nuevos elementos de reconocimiento selectivo para la monitorización de inmunosupresores en muestras sanguíneas

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 14-07-2022Cada año aumenta la demanda de trasplantes de órganos en todo el mundo, y según los datos del Observatorio Mundial de Donación y Trasplante y de la Organización Mundial de la Salud, en 2019 se realizaron más de 153,800 intervenciones en 84 países. El éxito del trasplante y la correcta recuperación del paciente dependen, en gran parte, de que se controlen en el paciente los niveles adecuados de medicamentos inmunosupresores (ISDs) tras el trasplante, los cuales presentan ventanas terapéuticas estrechas y grandes variaciones de absorción inter e intraindividuo (dando origen aniveles impredecibles en la sangre). Por ello, uno de los objetivos fundamentales de las autoridades de la Salud Pública es la monitorización farmacoterapéutica de estos medicamentos, para asegurar la máxima respuesta terapéutica con los mínimos efectos adversos. Actualmente, en los laboratorios clínicos se emplea mayoritariamente la cromatografía líquida acoplada a diferentes detectores (ej. matriz de diodos, fluorescencia o espectrometría de masas) para la determinación de ISDs. Si bien todas estas técnicas proporcionan métodos sensibles y reproducibles, requieren personal debidamente entrenado, en algunos casos su coste es elevado y, en ocasiones, se requieren etapas adicionales de extracción y derivatización, aumentando los tiempos de análisis y la irreproducibilidad del método. Además, estos métodos no son adecuados para la monitorización semicontinua en dispositivos de análisis in situ (POC, del inglés point-of care)o para el cribado de alto rendimiento...The demand for organ transplantation increases worldwide every year and, according to the Global Observatory on Donation and Transplantation and the World Health Organization, more than 153,800 transplants were performed in 84 countries in 2019. The success of transplantation and correct recovery of the patient depends, to a large extent, on the control of adequate levels of immunosuppressive drugs (ISDs) in blood, as they present narrow therapeutic windows and large inter- and intra-individual absorption variations (that may result in unpredictable levels). Therefore, one of the main objectives of Public Health authorities is to ensure the adequate pharmacotherapeutic monitoring of immunosuppressants in organ recipients to ensure a maximum therapeutic response with the minimum adverse effects. Currently, liquid chromatography coupled to different detectors (e.g. diode array, fluorescence or mass spectrometry) is widely applied for the control of ISDs in clinical laboratories. Although all these techniques allow the development of sensitive and reproducible analytical methods, they require properly trained personnel and, in most cases, complex sample pretreatment steps that increase irreproducibility, the analysis time and the cost per assay. Furthermore, these methods are not suitable for semicontinuous monitoring in point-of-care (POC) devices or for high-throughput screening...Fac. de Ciencias QuímicasTRUEunpu

Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids

Tacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter-and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micro motors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.Ministerio de Ciencia e InnovaciónComunidad de Madri

Optical Biosensors for Label-Free Detection of Small Molecules

Label-free optical biosensors are an intriguing option for the analyses of many analytes, as they offer several advantages such as high sensitivity, direct and real-time measurement in addition to multiplexing capabilities. However, development of label-free optical biosensors for small molecules can be challenging as most of them are not naturally chromogenic or fluorescent, and in some cases, the sensor response is related to the size of the analyte. To overcome some of the limitations associated with the analysis of biologically, pharmacologically, or environmentally relevant compounds of low molecular weight, recent advances in the field have improved the detection of these analytes using outstanding methodology, instrumentation, recognition elements, or immobilization strategies. In this review, we aim to introduce some of the latest developments in the field of label-free optical biosensors with the focus on applications with novel innovations to overcome the challenges related to small molecule detection. Optical label-free methods with different transduction schemes, including evanescent wave and optical fiber sensors, surface plasmon resonance, surface-enhanced Raman spectroscopy, and interferometry, using various biorecognition elements, such as antibodies, aptamers, enzymes, and bioinspired molecularly imprinted polymers, are reviewed

Molecular super-gluing: a straightforward tool for antibody labelling and its application to mycotoxin biosensing

Mycotoxins are low molecular weight toxic compounds, which can cause severe health problems in animals and humans. Immunoassays allow rapid, simple and cost-efective screening of mycotoxins. Sandwich assays with a direct readout provide great improvement in terms of selectivity and sensitivity, compared to the widely used competitive assay formats, for the analysis of low molecular weight molecules. In this work, we report a non-competitive fuorescence anti-immune complex (IC) immunoassay, based on the specifc recognition of HT-2 toxin with a pair of recombinant antibody fragments, namely antigen-binding fragment (Fab) (anti-HT-2 (10) Fab) and single-chain variable fragment (scFv) (anti-IC HT-2 (10) scFv). The SpyTag and SpyCatcher glue proteins were applied for the frst time as a bioconjugation tool for the analysis of mycotoxins. To this aim, a SpyTag-mScarlet-I (fuorescent protein) and scFv-SpyCatcher fusion proteins were constructed, produced and fused in situ during the assay by spontaneous Tag-Catcher binding. The assay showed an excellent sensitivity with an EC50 of 4.8±0.4 ng mL−1 and a dynamic range from 1.7±0.3 to 13±2 ng mL−1, an inter-day reproducibility of 8.5% and a high selectivity towards HT-2 toxin without cross-reactivity with other Fusarium toxins. The bioassay was applied to the analysis of the toxin in an oat reference material and in oat samples, with a LOD of 0.6 µg kg−1, and the results were validated by analysing a certifcate reference material and by HPLC–MS/MS

Biosensing Tacrolimus in Human Whole Blood by Using a Drug Receptor Fused to the Emerald Green Fluorescent Protein

Tacrolimus (FK506) is an immunosuppressant drug (ISD) used to prevent organ rejection after transplantation that exhibits a narrow therapeutic window and is subject to wide inter- and intra-individual pharmacokinetic fluctuations requiring careful monitoring. The immunosuppressive capacity of FK506 arises from the formation of a complex with immunophilin FKBP1A. This paper describes the use of FKBP1A as an alternative to common antibodies for biosensing purposes. Bioassays use recombinant FKBP1A fused to the emerald green fluorescent protein (FKBP1A–EmGFP). Samples containing the immunosuppressant are incubated with the recombinant protein, and free FKBP1A–EmGFP is captured by magnetic beads functionalized with FK506 to generate a fluorescence signal. Recombinant receptor–drug interaction is evaluated by using a quartz crystal microbalance and nuclear magnetic resonance. The limit of detection (3 ng mL–1) and dynamic range thus obtained (5–70 ng mL–1) fulfill therapeutic requirements. The assay is selective for other ISD usually coadministered with FK506 and allows the drug to be determined in human whole blood samples from organ transplant patients with results comparing favorably with those of an external laboratory

"Chem-game", el juego como estrategia para la dinamización del aprendizaje y la evaluación de conocimientos en Química General

Este proyecto pretende aplicar la "gamificación" en la enseñanza de la asignatura de Química General de primer curso del grado en Química para fomentar la formación, creatividad, compromiso y la capacidad de trabajo en equipo de los estudiantes

Optical Biosensors for Label-Free Detection of Small Molecules

Label-free optical biosensors are an intriguing option for the analyses of many analytes, as they offer several advantages such as high sensitivity, direct and real-time measurement in addition to multiplexing capabilities. However, development of label-free optical biosensors for small molecules can be challenging as most of them are not naturally chromogenic or fluorescent, and in some cases, the sensor response is related to the size of the analyte. To overcome some of the limitations associated with the analysis of biologically, pharmacologically, or environmentally relevant compounds of low molecular weight, recent advances in the field have improved the detection of these analytes using outstanding methodology, instrumentation, recognition elements, or immobilization strategies. In this review, we aim to introduce some of the latest developments in the field of label-free optical biosensors with the focus on applications with novel innovations to overcome the challenges related to small molecule detection. Optical label-free methods with different transduction schemes, including evanescent wave and optical fiber sensors, surface plasmon resonance, surface-enhanced Raman spectroscopy, and interferometry, using various biorecognition elements, such as antibodies, aptamers, enzymes, and bioinspired molecularly imprinted polymers, are reviewed

Magnetic Janus micromotors for fluorescence biosensing of tacrolimus in oral fluids

Tacrolimus (FK506) is a macrolide lactone immunosuppressive drug that is commonly used in transplanted patients to avoid organ rejection. FK506 exhibits high inter- and intra-patient pharmacokinetic variability, making monitoring necessary for organ graft survival. This work describes the development of a novel bioassay for monitoring FK506. The bioassay is based on using polycaprolactone-based (PCL) magnetic Janus micromotors and a recombinant chimera receptor that incorporates the immunophilin tacrolimus binding protein 1A (FKBP1A) tagged with Emerald Green Fluorescent Protein (EmGFP). The approach relies on a fluorescence competitive bioassay between the drug and the micromotors decorated with a carboxylated FK506 toward the specific site of the fluorescent immunophilin. The proposed homogeneous assay could be performed in a single step without washing steps to separate the unbound receptor. The proposed approach fits the therapeutic requirements, showing a limit of detection of 0.8 ng/mL and a wide dynamic range of up to 90 ng/mL. Assay selectivity was evaluated by measuring the competitive inhibition curves with other immunosuppressive drugs usually co-administered with FK506. The magnetic propulsion mechanism allows for efficient operation in raw samples without damaging the biological binding receptor (FKBP1A-EmGFP). The enhanced target recognition and micromixing strategies hold considerable potential for FK506 monitoring in practical clinical use.UCM for a predoctoral contractDepto. de Química AnalíticaFac. de Ciencias QuímicasTRUEpu

Sensitive rapid fluorescence polarization immunoassay for free mycophenolic acid determination in human serum and plasma

In this Article, we describe a fluorescence polarization immunoassay (FPIA) using a new label-near infrared fluorescent dye. The developed FPIA method was optimized for the rapid analysis of free mycophenolic acid (MPA) in plasma of transplanted patients. The approach is based on the fluorescence competitive assay between the target immunosuppressant and a novel emissive near-infrared fluorescent dye-tagged MPA and MPA-AO for the binding sites of the anti-MPA antibody. The fluorescent analogue of MPA exhibits emission at 654 nm upon excitation at 629 nm (λexcmax) and shows a good photochemical stability and a significant emission quantum yield (0.16) in phosphate buffer media. Free mycophenolic acid was isolated from blood or plasma samples using ultrafiltration prior to analysis. The sample was incubated for 20 min with 5 μg/mL of anti-MPA antibody and 1 nM of MPA-AO before the measurements. The developed FPIA displays a limit of detection of 0.8 ng/mL (10% binding inhibition) and a dynamic range of 1.7−39 ng/mL (20%−80% binding inhibition) in a PBST buffer, fitting the therapeutic requirements. The immunoassay selectivity was evaluated by measuring the cross-reactivity to other immunosuppressive drugs administered in combination with MPA (cyclosporin A and tacrolimus), as well as for the metabolite MPA glucuronide. The assay has been successfully applied to the analysis of free MPA in the blood of a heart-transplanted patient after oral administration of both mycophenolate mofetil (MMF) and tacrolimus, and the results have been compared with those obtained by rapid-resolution liquid chromatography with diode array detection (RRLC-DAD).Ministerio de Economía, Comercio y Empresa (España)Depto. de Química AnalíticaDepto. de Química OrgánicaFac. de Ciencias QuímicasFALSEunpu