Paving the way to point of care (POC) devices for SARS-CoV-2 detection

In this work we present a powerful, affordable, and portable biosensor to develop Point of care (POC) SARS-CoV-2 virus detection. It is constructed from a fast, low cost, portable and electronically automatized potentiostat that controls the potential applied to a disposable screen-printed electrochemical platform and the current response. The potentiostat was designed to get the best signal-to-noise ratio, a very simple user interface offering the possibility to be used by any device (computer, mobile phone or tablet), to have a small and portable size, and a cheap manufacturing cost. Furthermore, the device includes as main components, a data acquisition board, a controller board and a hybridization chamber with a final size of 10 × 8 × 4 cm. The device has been tested by detecting specific SARS-CoV-2 virus sequences, reaching a detection limit of 22.1 fM. Results agree well with those obtained using a conventional potentiostat, which validate the device and pave the way to the development of POC biosensors. In this sense, the device has finally applied to directly detect the presence of the virus in nasopharyngeal samples of COVID-19 patients and results confirm its utility for the rapid detection infected samples avoiding any amplification process.This work has been financially supported by the Spanish Ministry of Economy and Competitiveness (PID2020-116728RB-I00, CTQ2015-71955-REDT (ELECTROBIONET)) and Community of Madrid (TRANSNANOAVANSENS, S2018/NMT-4349). Authors also acknowledge REACT EU NANOCOV-CM project. We acknowledge the service from the MiNa Laboratory at IMN, and funding from Community of Madrid (project S2018/NMT-4291 TEC2SPACE), MINECO (project CSIC13-4E-1794) and EU (FEDER, FSE)”. RdC gratefully thanks support from Fundación IMDEA, UAM and Banco Santander (fondo supera 2020, convocatoria CRUE–CSIC–SANTANDER, project with reference 10.01.03.02.41).Peer reviewe

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 μM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification processPID2020-116728RB-I00, PID2020-116661RB-I00, PID2020-119352RB-I00, PDC2021-120782-C2, PID2022-138908NB-C31, CTQ2015-71955-REDT, S2018/NMT-434

CASCADE: Naked eye-detection of SARS-CoV-2 using Cas13a and gold nanoparticles

The COVID-19 pandemic has brought to light the need for fast and sensitive detection methods to prevent the spread of pathogens. The scientific community is making a great effort to design new molecular detection methods suitable for fast point-of-care applications. In this regard, a variety of approaches have been developed or optimized, including isothermal amplification of viral nucleic acids, CRISPR-mediated target recognition, and read-out systems based on nanomaterials. Herein, we present CASCADE (CRISPR/CAS-based Colorimetric nucleic Acid DEtection), a sensing system for fast and specific naked-eye detection of SARS-CoV-2 RNA. In this approach, viral RNA is recognized by the LwaCas13a CRISPR protein, which activates its collateral RNase activity. Upon target recognition, Cas13a cleaves ssRNA oligonucleotides conjugated to gold nanoparticles (AuNPs), thus inducing their colloidal aggregation, which can be easily visualized. After an exhaustive optimization of functionalized AuNPs, CASCADE can detect picomolar concentrations of SARS-CoV-2 RNA. This sensitivity is further increased to low femtomolar (3 fM) and even attomolar (40 aM) ranges when CASCADE is coupled to RPA or NASBA isothermal nucleic acid amplification, respectively. We finally demonstrate that CASCADE succeeds in detecting SARS-CoV-2 in clinical samples from nasopharyngeal swabs. In conclusion, CASCADE is a fast and versatile RNA biosensor that can be coupled to different isothermal nucleic acid amplification methods for naked-eye diagnosis of infectious diseases.This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2017-87305-R, PID2020-119352RB-I00), Instituto de Salud Carlos III (FONDO-COVID19:COV20/00144 and COV20/00122) and Madrid Regional Government (NANOCOV-CM and IND2017/IND7809). C.E-N, M. L-V and C.R-D thank Madrid Regional Government for the pre-doctoral Grants (PEJD-2017-PRE/BMD-3730, PEJD-2018-PRE/IND-9584 and PEJD-2017-PRE/IND-4438). P.M.R thanks the Ministry of Economy, Industry and competitiveness of Spain for the FPI fellowship (BES-2017.082521). IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centres of Excellence in R&D (MINECO, Grant SEV-2016-0686, CEX2020-001039-S).Peer reviewe

Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing

The development of DNA-sensing platforms based on new synthetized Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures (AuNs), as a new pathway to develop a selective and sensitive methodology for SARS-CoV-2 detection is presented. A mixture of gold nanoparticles and gold nanotriangles have been synthetized to modify disposable electrodes that act as an enhanced nanostructured electrochemical surface for DNA probe immobilization. On the other hand, modified carbon nanodots prepared a la carte to contain Methylene Blue (MB-CDs) are used as electrochemical indicators of the hybridization event. These MB-CDs, due to their structure, are able to interact differently with double and single-stranded DNA molecules. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected in a straightforward way and rapidly with a detection limit of 2.00 aM. Moreover, this platform allows the detection of the SARS-CoV-2 sequence in the presence of other viruses, and also a single nucleotide polymorphism (SNPs). The developed approach has been tested directly on RNA obtained from nasopharyngeal samples from COVID-19 patients, avoiding any amplification process. The results agree well with those obtained by RT-qPCR or reverse transcription quantitative polymerase chain reaction technique.We acknowledge the support from the Comunidad de Madrid (TRANSNANOAVANSENS-CM, S2018/NMT-4349, NANOCOV-CM, SI3/PJI/2021–00341) and Ministerio de economia y competitividad de España (PID2020–116728RB-100, CTQ2015–71955-REDT (ELECTROBIONET)). IMDEA Nanociencia acknowledges support from the Programme for Centres of Excellence in R&D ‘Severo Ochoa’ (CEX2020–001039-S, MINECO). Authors also acknowledge REACT EU NANOCOV-CM project. RdC acknowledges support from Fundación IMDEA Nanociencia, Banco Santander, UAM (convocatoria CRUE- SANTANDER-CSIC, reference 10.01.03.02.41).Peer reviewe

Terapias y diagnósticos basados en ncRNAs y nanotecnologías: reprogramando células cancerígenas y tratamientos multimodales

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología. Fecha de Lectura: 30-03-2023Esta tesis tiene embargado el acceso al texto completo hasta el 30-09-2024Cancer is the second cause of death worldwide, with 9.6 million deaths registered in 2018, despite the enormous efforts made by the scientific community 1. Several factors contribute to the development of this disease, including those related to genetic and epigenetic information. Within the latter case, it is worth mentioning the role of non-coding RNAs (ncRNAs), which are dysregulated in many pathologies, including cancer. For this reason, restoring the levels of crucial ncRNAs, such as microRNAs (ca. 22 nts) or lncRNAs (>200 nts), could restore the normal behavior of cells or increase their sensitivity to drugs, preventing resistances and relapses 2. However, oligonucleotide-based therapies present challenges, such as reduced tissue or cell selectivity, poor cell internalization, and low stability. On the other hand, chemotherapeutic drugs present poor aqueous solubility and affect healthy cells. To overcome these limitations, nanoformulations could be employed 3. In this sense, in this doctoral thesis, different nanoparticles for oligonucleotide delivery were used to regulate the expression levels of microRNA or lncRNA in combination with chemotherapeutic agents. Precisely, a cocktail of tumor suppressor microRNAs and the chemotherapeutic agent SN38 have been used to treat different types of cancers (e.g., uveal melanoma, lung, and breast cancer). This therapy was delivered by gold or magnetic nanoparticles. Regarding the lncRNA approach, siRNAs were designed to target oncogenic lncRNA such as MALAT1 or PVT1 to restore their normal levels in breast and colon cancer cells. Promising results were achieved in reducing tumorigenesis features with the combination of siRNAs against lncRNA and chemotherapeutic drugs. Regarding cancer diagnosis, modified gold nanoparticles were developed to detect dysregulated microRNAs or gene mutations. MicroRNAs were detected through a fluorescence gold nanoparticle sensor named nanoflares. Gene mutations (e.g., GNAQ or BRAF) were detected with nanoflares sensors and through a lateral flow assay (LFA) system coupled with a previous amplification method. In summary, in this thesis, different nanoformulations were investigated to develop diagnosis methods and therapeutic approaches based on ncRNA for cancerEl cáncer es la segunda causa de muerte en el mundo, con 9,6 millones de muertes registradas en 2018 a pesar del enorme esfuerzo hecho por la comunidad científica 1. Hay varios factores que contribuyen al desarrollo de la enfermedad, entre los que se incluyen aquellos relacionados con la genética y la epigenética. En referencia a estos últimos, vale la pena mencionar el papel de los ARN no codificantes (ncARN), los cuales están desregulados en muchas patologías, incluido en el cáncer. Por esta razón, restaurar los niveles claves de ncARNs, como los microARNs (alrededor de 22 nts) o lncARNs (>200 nts) puede restaurar un comportamiento más sano de las células o aumentar su sensibilidad a fármacos, por lo tanto reducir la resistencia a fármacos y recaídas de la enfermedad 2. Sin embargo, las terapias con oligonucleótidos presentan una serie de desafíos como por ejemplo una baja selectividad hacia los tejidos deseados, pobre internalización celular y baja estabilidad. Por otro lado, los fármacos quimioterapéuticos tienen baja solubilidad en agua y afectan a células sanas. Para superar estas limitaciones se emplean nanoformulaciones 3. En este sentido, en esta tesis doctoral se han usado diferentes nanopartículas para transportar oligonucleótidos que regulan los niveles de expresión de microARNs o lncARNs, en combinación con agentes quimioterapéuticos. En detalle, una mezcla de microARNs supresores de tumores combinados con el agente quimioterapéutico SN38 fue utilizado para tratar diferentes tipos de cáncer (por ejemplo el melanoma de úvea, cáncer de pulmón o cáncer de mama). Esta terapia fue transportada por nanopartículas de oro o magnéticas. Con referencia a lo aproximación de los lncARNs, se diseñaron siARNs para afectar los niveles de lncARNs oncogénicos como MALAT1 o PVT1 en células de cáncer de mama o colon. Se obtuvieron resultados prometedores reduciendo las características tumorales con la combinación de siARNs y fármacos quimioterapéuticos. Con referencia al diagnóstico del cáncer, se desarrollaron sistemas basados en nanopartículas de oro modificas para detectar microARNs o mutaciones de genes. Los microARNs fueron detectados gracias a sensores fluorescentes basados en nanopartículas de oro conocidos como nanoflares. Las mutaciones de genes (como GNAQ o BRAF) fueron detectas gracias a sensores tipo nanoflare y también con ensayos de flujo lateral acoplados a una previa amplificación. En resumen, en esta tesis, diferentes nanoformulaciones han sido investigadas para desarrollar métodos de diagnóstico y aproximaciones terapéuticas basadas en ncARNs contra el cánce

The Role of LncRNAs in Uveal Melanoma

Uveal melanoma (UM) is an intraocular cancer tumor with high metastatic risk. It is considered a rare disease, but 90% of affected patients die within 15 years. Non-coding elements (ncRNAs) such as long non-coding RNAs (lncRNAs) have a crucial role in cellular homeostasis maintenance, taking part in many critical cellular pathways. Their deregulation, therefore, contributes to the induction of cancer and neurodegenerative and metabolic diseases. In cancer, lncRNAs are implicated in apoptosis evasion, proliferation, invasion, drug resistance, and other roles because they affect tumor suppressor genes and oncogenes. For these reasons, lncRNAs are promising targets in personalized medicine and can be used as biomarkers for diseases including UM

Reprogramming Cells for Synergistic Combination Therapy with Nanotherapeutics against Uveal Melanoma

Uveal melanoma (UM) is the most common primary intraocular malignant tumor in adults and around half of the patients develop metastasis and die shortly after because of the lack of effective therapies for metastatic UM. Consequently, new therapeutic approaches to this disease are welcome. In this regard, microRNAs have been shown to have a key role in neoplasia progression and have the potential to be used as therapeutic tools. In addition, in different cancers including UM, a particular microRNA signature appears that is different from healthy cells. Thus, restoring the regular levels of microRNAs could restore the normal behavior of cells. In this study, four microRNAs downregulated in UM have been chosen to reprogram cancer cells, to promote cell death or increase their sensitivity to the chemotherapeutic SN38. Furthermore, to improve the internalization, stability and/or solubility of the therapeutic molecules employed in this approach, gold nanoparticles (AuNPs) were used as carriers. Remarkably, this study found a synergistic effect when the four oligonucleotides were employed and when the chemotherapeutic drug was added

Boron Dipyrromethene (BODIPY) as Electron-Withdrawing Group in Asymmetric Copper-Catalyzed [3+2] Cycloadditions for the Synthesis of Pyrrolidine-Based Biological Sensors

This is the peer reviewed version of the following article: Rigotti, T., Asenjo‐Pascual, J., Martín‐Somer, A., Milán Rois, P., Cordani, M., Díaz‐Tendero, S., ... & Alemán. Boron Dipyrromethene (BODIPY) as Electron‐Withdrawing Group in Asymmetric Copper‐Catalyzed [3+ 2] Cycloadditions for the Synthesis of Pyrrolidine‐Based Biological Sensors. Advanced Synthesis & Catalysis Volume 362, Issue 6, 17 March 2020, Pages 1345-1355 , which has been published in final form at https://doi.org/10.1002/adsc.201901465. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsIn this work, we describe the use of Boron Dipyrromethene (BODIPY) as electron-withdrawing group for activation of double bonds in asymmetric copper-catalyzed [3+2] cycloaddition reactions with azomethine ylides. The reactions take place under smooth conditions and with high enantiomeric excess for a large number of different substituents, pointing out the high activation of the alkene by using a boron dipyrromethene as electron-withdrawing group. Experimental, theoretical studies and comparison with other common electron-withdrawing groups in asymmetric copper-catalyzed [3+2] cycloadditions show the reasons of the different reactivity of the boron dipyrromethene derivatives, which can be exploited as a useful activating group in asymmetric catalysis. Additional experiments show that the so obtained pyrrolidines can be employed as biocompatible biosensors, which can be located in the endosomal compartments and do not present toxicity in three cell linesThis work was supported by the Spanish Government (RTI2018‐095038‐B‐I00, CTQ2016‐76061‐P, SAF2017‐87305‐R), Comunidad de Madrid (IND2017/IND‐7809), and co‐financed by European Structural and Investment Fund. We acknowledge the generous allocation of computing time at the CCC (UAM). Financial support from the Spanish Ministry of Economy and Competitiveness, through the ‘‘Maria de Maeztu’’ Program of Excellence in R&D (MDM‐2014‐0377), is also acknowledged. Asociación Española Contra el Cáncer, and IMDEA Nanociencia acknowledge support from the ′Severo Ochoa′ Programme for Centres of Excellence in R&D (MINECO, Grant SEV‐2016‐0686). P.M.R thanks the Ministry of Economy, Industry and competitiveness of Spain for the FPI grant (BES‐2017.082521). A.M.S. thanks CAM for a postdoctoral contract (2016‐T2/IND‐1660

Water Soluble Iron-Based Coordination Trimers as Synergistic Adjuvants for Pancreatic Cancer

International audiencePancreatic cancer is a usually fatal disease that needs innovative therapeutic approaches since the current treatments are poorly effective. In this study, based on cell lines, triazole-based coordination trimers made with soluble Fe(II) in an aqueous media were explored for the first time as adjuvant agents for the treatment of this condition. These coordination complexes were effective at relatively high concentrations and led to an increase in reactive oxygen species (ROS) in two pancreatic cancer cell lines, PANC-1 and BXPC-3, and this effect was accompanied by a significant reduction in cell viability in the presence of gemcitabine (GEM). Importantly, the tested compounds enhanced the effect of GEM, an approved drug for pancreatic cancer, through apoptosis induction and downregulation of the mTOR pathway. Although further evaluation in animal-based models of pancreatic cancer is needed, these results open novel avenues for exploring these iron-based materials in biomedicine in general and in pancreatic cancer treatment

Smart Modification on Magnetic Nanoparticles Dramatically Enhances Their Therapeutic Properties

Magnetic nanoparticles (MNP) are employed as nanocarriers and in magnetic hyperthermia (MH) for the treatment of cancers. Herein, a smart drug delivery system composed of MNP functionalized with the cytotoxic drug gemcitabine (MNP-GEM) has been thoroughly evaluated. The linker employed is based on a disulfide bond and allows the controlled release of GEM under a highly reducing environment, which is frequently present in the cytoplasm of tumor cells. The stability, MH, and the interaction with plasma proteins of the nanoparticles are evaluated, highlighting their great potential for biological applications. Their cytotoxicity is assessed in three pancreatic cancer cell lines with different sensitivity to GEM, including the generation of reactive oxygen species (ROS), the effects on the cell cycle, and the mechanisms of cell death involved. Remarkably, the proposed nanocarrier is better internalized than unmodified nanoparticles, and it is particularly effective in PANC-1 cells, resistant to GEM, but not in non-tumoral keratinocytes. Additionally, its combination with MH produces a synergistic cytotoxic effect in all cancer cell lines tested. In conclusion, MNP-GEM presents a promising potential for treating pancreatic cancer, due to multiple parameters, such as reduced binding to plasma proteins, increased internalization, and synergistic activity when combined with MH