Xeno-free bioengineered human skeletal muscle tissue using human platelet lysate-based hydrogels

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications.The authors thank the technical support of MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS 'NANBIOSIS' from CIBER-BBN at IBEC. We would also like to thank the muscle team from the Biosensors for Bioengineering group for their feedback in the review process of this manuscript. Human immortalized muscle satellite stem cells used in this study were kindly provided by Dr Bénédicte Chazaud (Institut NeuroMyoGène (INMG), Lyon, France). This project received financial support from European Research Council program Grant ERC-StG-DAMOC: 714317 (J R-A), European Commission under FET-open program BLOC Project: GA- 863037 (J R-A), Spanish Ministry of Economy and Competitiveness, through the 'Severo Ochoa' Program for Centres of Excellence in R&D: SEV-2016–2019, Spanish Ministry of Economy and Competitiveness: 'Retos de investigación: Proyectos I+D+i': TEC2017-83716-C2-2-R (J R-A), CERCA Programme/Generalitat de Catalunya: 2017-SGR-1079 (J R-A), and Fundación Bancaria 'la Caixa'- Obra Social 'la Caixa': project IBEC-La Caixa Healthy Ageing (J R-A). The authors also acknowledge the European Union's Horizon 2020 research and innovation program under European Research Council Grant Agreement 772817 and Twinning Grant Agreement No. 810850—Achilles. Fundação para a Ciência e a Tecnologia (FCT) for CEECIND/01375/2017 (M G-F) and 2020.03410.CEECIND (R M A D)

Interdigitated μ-electrodes for development of an impedimetric immunosensor for atrazine detection.

This contribution describes the development of an impedimetric immunosensor for atrazine detection. This immunosensor is based on the use of interdigitated metallic μ-electrodes (IDμEs) The method described in this work does not use any redox mediator and relies on the direct detection of immunochemical competitive reaction between the pesticide and a haptenized-protein immobilized on interdigitated μ-electrodes for the specific antibody. The immunoreagents used were specifically developed to detect atrazine. The immunochemical detection of this pesticide is achieved without using any label. The immunosensor shows a limit of detection of 8.34±1.37 μg L-1, witch is lower than the Maximun Residue Level (MRL) (50μg L-1)established by EU (European Union)for residues of atrazine as herbicide in the wine grapes and other foodstuff products.Peer ReviewedPostprint (published version

Immunosensor impedimetrico para la detección de pesticidas

En este trabajo describimos un immunosensor impedimétrico para la detección de pesticidas. Para demostrar dicho sensor hemos utilizado atrazina, como pesticida de test. Este sensor está basado en el uso de μ-electrodos interdigitados así como en reactivos específicamente desarrollados para la detección de este pesticida. Los anticuerpos utilizados no incluyen ningún tipo de etiqueta. Así mismo, el sensor no incluye ningún tipo de par redox que amplifique la señal. La detección immunoquímica de atrazina se alcanza mediante una reacción competitiva entre el antígeno tapizado y el pesticida por una pequeña cantidad de anticuerpo. Los cambios en la impedancia producidos por la inclusión de los bioreactivos son interpretados utilizando un circuito equivalente, el cual representa el sistema de manera fiable. La detección se monitoriza a partir de medidas impedimétricas diferenciales en un amplio espectro de frecuencia. El immunosensor muestra límites de detección en el rango de pocos ppb's, lo cual está muy por debajo del Maximum Residue Level (MRL) (50 μg L-1) establecido por la Unión Europea para los residuos de atrazina en uvas de vino así como en otros productos alimenticios. Aunque en este trabajo el immunosensor se ha demostrado para la atrazina, otros pesticidas podrían detectarse mediante este método siempre que se utilicen los reactivos adecuados.Peer ReviewedPostprint (author’s final draft

Integrated Bioluminescent Immunoassays for High-Throughput Sampling and Continuous Monitoring of Cytokines

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an &gt;80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.</p

An Immunoassay for Dibutyl Phthalate Based on Direct Hapten Linkage to the Polystyrene Surface of Microtiter Plates

BACKGROUND: Dibutyl phthalate (DBP) is predominantly used as a plasticizer inplastics to make them flexible. Extensive use of phthalates in both industrial processes and other consumer products has resulted in the ubiquitous presence of phthalates in the environment. In order to better determine the level of pollution in the environment and evaluate the potential adverse effects of exposure to DBP, immunoassay for DBP was developed. METHODOLOGY/PRINCIPAL FINDINGS: A monoclonal antibody specific to DBP was produced from a stable hybridoma cell line generated by lymphocyte hybridoma technique. An indirect competitive enzyme-linked immunosorbent assay (icELISA) employing direct coating of hapten on polystyrene microtiter plates was established for the detection of DBP. Polystyrene surface was first oxidized by permanganate in dilute sulfuric acid to generate carboxyl groups. Then dibutyl 4-aminophthalate, which is an analogue of DBP, was covalently linked to the carboxyl groups of polystyrene surface with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). Compared with conjugate coated format (IC(50)=106 ng/mL), the direct hapten coated format (IC(50)=14.6 ng/mL) improved assay sensitivity after careful optimization of assay conditions. The average recovery of DBP from spiked water sample was 104.4% and the average coefficient of variation was 9.95%. Good agreement of the results obtained by the hapten coated icELISA and gas chromatography-mass spectrometry further confirmed the reliability and accuracy of the icELISA for the detection of DBP in certain plastic and cosmetic samples. CONCLUSIONS/SIGNIFICANCE: The stable and efficient hybridoma cell line obtained is an unlimited source of sensitive and specific antibody to DBP. The hapten coated format is proposed as generally applicable because the carboxyl groups on modified microtiter plate surface enables stable immobilization of aminated or hydroxylated hapten with EDC. The developed hapten coated icELISA can be used as a convenient quantitative tool for the sensitive and accurate monitoring DBP in water, plastic and cosmetic samples

Haptenos, usos y método inmunoquímico para la detección de bromopropilato

Haptenos, usos y método inmunoquímico para la detección de bromopropilato. La presente invención se refiere a varios haptenos derivados de bromopropilato (BP), sus usos para la síntesis de inmunoreactivos así como un método inmunoquímico para la detección y/o cuantificación de bromopropilato (BP) y sus respectivos kits. La estructura química de los haptenos empleados, tanto para la inmovilización en el soporte sólido como para la inmunización de animales para obtener los respectivos anticuerpos policlonales, se ha diseñado para maximizar el reconocimiento del grupo bisbromofenil del BP por parte de los anticuerpos generados. El método de la presente invención se refiere a un análisis inmunoquímico indirecto capaz de detectar BP con un umbral de 0.14 μg·L-1 y pesticidas relacionados que contienen un grupo bis-halofenil en su estructura. Además, el BP se puede analizar directamente en muestras de vino.Consejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Haptenos, usos y método inmunoquímico para la detección de bromopropilato

[EN] The invention relates to various haptens derived from bromopropylate (BP), the uses thereof for the synthesis of immunoreagents and an immunochemical method for bromopropylate (BP) detection andlor quantification, as well as the respective kits therefor. The chemical structure of the haptens employed, both for immobilisation on the solid support and for immunisation of animals to obtain the respective polyclonal antibodies, has been designed to maximise recognition of the bisbromophenyl group of the BP by the antibodies generated. The method of the invention relates to indirect immunochemical analysis capable of detecting BP with a threshold of 0.14 µgL-1 and related pesticides containing a bis-halophenyl group in the structure thereof. Furthermore, BP can be analysed directly in wine samples.[ES] La presente invención se refiere a varios haptenos derivados de bromopropilato(BP), sus usos para la síntesis de inmunoreactivos así como un método inmunoquímico para la detección y/o cuantificación de bromopropilato (BP) y sus respectivos kits. La estructura química de los haptenos empleados, tanto para la inmovilización en el soporte sólido como para la inmunización de animales para obtener los respectivos anticuerpos policlonales, se ha diseñado para maximizar el reconocimiento del grupo bis-bromofenil del BP por parte de los anticuerpos generados. El método de la presente invención se refiere a un análisis inmunoquímico indirecto capaz de detectar BP con un umbral de 0.14 µg-L-1 y pesticidas relacionados que contienen un grupo bis-halofenil en su estructura. Además, el BP se puede analizar directamente en muestras de vino.Peer reviewedConsejo Superior de Investigaciones Científicas (España)A1 Solicitud de patente con informe sobre el estado de la técnic

Development of an enzyme-linked immunosorbent assay for the determination of the linear alkylbenzene sulfonates and long-chain sulfophenyl carboxylates using antibodies generated by pseudoheterologous immunization

11 pages, 5 tables, 7 figures.-- PMID: 16383312 [PubMed].-- Available online Nov 25, 2005.Supporting information available at: http://pubs.acs.org/doi/suppl/10.1021/ac051141sELISA methods have been developed for screening contamination of water resources by linear alkyl benzene sulfonates (LAS) or the most immediate degradation products, the long chain sulfophenyl carboxylates, SPCs. The assay uses antibodies raised through pseudoheterologous immunization strategies using an equimolar mixture of two immunogens (SFA−KLH and 13C13-SPC−KLH) prepared by coupling N-(4-alkylphenyl)sulfonyl-3-aminopropanoic acid (SFA) and p-(1-carboxy-13-tridecyl)phenylsulfonic acid (13C13-SPC) to keyhole limpet hemocyanin (KLH). The immunizing haptens have been designed to address recognition versus two different epitopes of the molecule. The SFA hapten maximizes recognition of the alkyl moiety while preserving the complexity of the different alkyl chains present in the LAS technical mixture. The 13C13-SPC hapten addresses recognition of the common and highly antigenic phenylsulfonic group. The antisera raised using this strategy have been shown to be superior to those obtained through homologous immunization procedures using a single substance. By using an indirect ELISA format, LAS and long-chain SPCs can be detected down to 1.8 and 0.2 μg L-1, respectively. Coefficients of variation of 6 and 12% within and between assays, respectively, demonstrate immunoassay reproducibility. The assay can be used in media with a wide range of pH and ionic strength values. Preliminary experiments performed to assess matrix effects have demonstrated the potential applicability of the method as a screening tool to assess contamination by these types of surfactants in natural water samples.This work has been supported by the Spanish Ministry of Science and Technology (Contract numbers ALG2002-04635-C04-03 and TEC2004-0121-E), the European Community (IST-2003-508774) and by the Department of Universities, Research and Information Society of the Generalitat of Catalonia (expedient 00207).Peer reviewe

Sistema y procedimiento multianalítico basado en mediciones impedimétricas

[ES] La invención describe un sistema para la detección y/o cuantificación simultánea de varios analitos en una muestra o bien para la detección y/o cuantificación simultánea de un analito en varias muestras, que comprende: un chip (10) multielectrodo que comprende un conjunto de microelectrodos (lla, 11 b, 11c, lId); una celda (20) de conducto único, que comprende una ranura (21 ) que aloja el chip (10) multielectrodo y un conducto (22) por el que una muestra fluida puede circular secuencialmente por cada uno de los microelectrodos[EN] The invention describes a system for the simultaneous detection and/or quantification of several analytes in a sample or for the simultaneous detection and/or quantification of an analyte in several samples, comprising: a multi-electrode chip (10) comprising a set ofmicroelectro des (1 la, 11 b, llc, lId); a single-duct cell (20) comprising a slot (21) containing the multi-electrode chip (10) and a duct (22) through which a fluid sample can flow sequentially over each of the microelectro des (lla, 11 b, llc, lId) of a multi-electrode chip (10) when it is contained in the slot (21); and a multi-duct cell (30) comprising a slot (31) containing the multi-electrode chip (10) and several separate ducts (32a, 32b, 32c, 32d) through which several samples can flow separately over each electro de (lla, 11 b, 11c, lId) ofthe multi-electrode chip (10).Peer reviewedConsejo Superior de Investigaciones Científicas España)A1 Solicitud de patente con informe sobre el estado de la técnic