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

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

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