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 process.

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.

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.

Amplification-free detection of SARS-CoV-2 using gold nanotriangles functionalized with oligonucleotides.

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipment.

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.

Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection.

We present a fast, reliable and easy to scale-up colorimetric sensor based on gold nanoparticles (AuNPs) to detect the sequences coding for the RdRp, E, and S proteins of SARS-CoV-2. The optimization of the system (so-called "the sensor") includes the evaluation of different sizes of nanoparticles, sequences of oligonucleotides and buffers. It is stable for months without any noticeable decrease in its activity, allowing the detection of SARS-CoV-2 sequences by the naked eye in 15 min. The efficiency and selectivity of detection, in terms of significative colorimetric changes in the solution upon target recognition, are qualitatively (visually) and quantitatively (absorbance measurements) assessed using synthetic samples and samples derived from infected cells and patients. Furthermore, an easy and affordable amplification approach is implemented to increase the system's sensitivity for detecting high and medium viral loads (≥103 - 104 viral RNA copies/µl) in patient samples. The whole process (amplification and detection) takes 2.5 h. Due to the ease of use, stability and minimum equipment requirements, the proposed approach can be a valuable tool for the detection of SARS-CoV-2 at facilities with limited resources.

Analysis of the diagnostic accuracy of rapid antigenic tests for detection of SARS-CoV-2 in hospital outbreak situation.

The reverse transcriptase polymerase chain reaction (RT-PCR) continues to be the reference diagnostic method for the confirmation of COVID-19 cases; however, rapid antigen detection tests (RADT) have recently been developed. The purpose of the study is to assess the performance of rapid antigen-based COVID-19 testing in the context of hospital outbreaks. This was an observational, cross-sectional study. The study period was from October 2020 to January 2021. The "Panbio COVID-19 AG" RADT (Abbott) was performed and TaqPath COVID-19 test RT-PCR. The samples were obtained from hospitalised patients in suspected outbreak situations at the Ramón y Cajal Hospital. A hospital outbreak was defined as the presence of 3 or more epidemiologically linked cases. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RADT were calculated using RT-PCR as a reference. A total of 17 hospital outbreaks were detected in 11 hospital units during the study period, in which 34 RT-PCR and RADT screenings were performed. We obtained 541 samples, which were analysed with RT-PCR and a further 541 analysed with RADT. Six RADT tests gave conflicting results with the RT-PCR, 5 of them with a negative RADT and positive RT-PCR and one with positive RADT and a negative RT-PCR. The sensitivity of the RADT was 83.3% (65.3-94.4%) and the specificity was 99.8% (98.9-100%). The PPV was 96.2% (80.4-99.9%) and the NPV was 99% (97.7-99.7%). The RADT shows good diagnostic performance in patients on non-COVID-19 hospital wards, in the context of an outbreak.

Emergence and Spread of B.1.1.7 Lineage in Primary Care and Clinical Impact in the Morbi-Mortality among Hospitalized Patients in Madrid, Spain.

In December 2020, UK authorities warned of the rapid spread of a new SARS-CoV-2 variant, belonging to the B.1.1.7 lineage, known as the Alpha variant. This variant is characterized by 17 mutations and 3 deletions. The deletion 69-70 in the spike protein can be detected by commercial platforms, allowing its real-time spread to be known. From the last days of December 2020 and over 4 months, all respiratory samples with a positive result for SARS-CoV-2 from patients treated in primary care and the emergency department were screened to detect this variant based on the strategy S gene target failure (SGTF). The first cases were detected during week 53 (2020) and reached >90% of all cases during weeks 15-16 (2021). During this period, the B.1.1.7/SGTF variant spread at a rapid and constant replacement rate of around 30-36%. The probability of intensive care unit admission was twice higher among patients infected by the B.1.1.7/SGTF variant, but there were no differences in death rate. During the peak of the third pandemic wave, this variant was not the most prevalent, and it became dominant when this wave was declining. Our results confirm that the B.1.1.7/SGTF variant displaced other SARS-CoV-2 variants in our healthcare area in 4 months. This displacement has led to an increase in the burden of disease.