State-of-the-art colloidal particles and unique interfaces-based SARS-CoV-2 detection methods and COVID-19 diagnosis

In March 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-based infections were declared ‘COVID-19 pandemic’ by the World Health Organization. Pandemic raised the necessity to design and develop genuine and sensitive tests for precise specific SARS-CoV-2 infections detection. Nanotechnological methods offer new ways to fight COVID-19. Nanomaterials are ideal for unique sensor platforms because of their chemically versatile properties and they are easy to manufacture. In this context, selected examples for integrating nanomaterials and distinct biosensor platforms are given to detect SARS-CoV-2 biological materials and COVID-19 biomarkers, giving researchers and scientists more goals and a better forecast to design more relevant and novel sensor arrays for COVID-19 diagnosis.     

Ultrasensitive Detection of COVID-19 Causative Virus (SARS-CoV-2) Spike Protein Using Laser Induced Graphene Field-Effect Transistor

COVID-19 is a highly contagious human infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and the war with the virus is still underway. Since no specific drugs have been made available yet and there is an imbalance between supply and demand for vaccines, early diagnosis and isolation are essential to control the outbreak. Current nucleic acid testing methods require high sample quality and laboratory conditions, which cannot meet flexible applications. Here, we report a laser-induced graphene field-effect transistor (LIG-FET) for detecting SARS-CoV-2. The FET was manufactured by different reduction degree LIG, with an oyster reef-like porous graphene channel to enrich the binding point between the virus protein and sensing area. After immobilizing specific antibodies in the channel, the FET can detect the SARS-CoV-2 spike protein in 15 min at a concentration of 1 pg/mL in phosphate-buffered saline (PBS) and 1 ng/mL in human serum. In addition, the sensor shows great specificity to the spike protein of SARS-CoV-2. Our sensors can realize fast production for COVID-19 rapid testing, as each LIG-FET can be fabricated by a laser platform in seconds. It is the first time that LIG has realized a virus sensing FET without any sample pretreatment or labeling, which paves the way for low-cost and rapid detection of COVID-19.     

Multiplex metal-detection based assay (MMDA) for COVID-19 diagnosis and identification of disease severity biomarkers

The ongoing COVID-19 pandemic caused by SARS-CoV-2 highlights the urgent need to develop sensitive methods for diagnosis and prognosis. To achieve this, multidimensional detection of SARS-CoV-2 related parameters including virus loads, immune response, and inflammation factors is crucial. Herein, by using metal-tagged antibodies as reporting probes, we developed a multiplex metal-detection based assay (MMDA) method as a general multiplex assay strategy for biofluids. This strategy provides extremely high multiplexing capability (theoretically over 100) compared with other reported biofluid assay methods. As a proof-of-concept, MMDA was used for serologic profiling of anti-SARS-CoV-2 antibodies. The MMDA exhibits significantly higher sensitivity and specificity than ELISA for the detection of anti-SARS-CoV-2 antibodies. By integrating the high dimensional data exploration/visualization tool (tSNE) and machine learning algorithms with in-depth analysis of multiplex data, we classified COVID-19 patients into different subgroups based on their distinct antibody landscape. We unbiasedly identified anti-SARS-CoV-2-nucleocapsid IgG and IgA as the most potently induced types of antibodies for COVID-19 diagnosis, and anti-SARS-CoV-2-spike IgA as a biomarker for disease severity stratification. MMDA represents a more accurate method for the diagnosis and disease severity stratification of the ongoing COVID-19 pandemic, as well as for biomarker discovery of other diseases.

A MMDA platform is developed by using metal-tagged antibodies as reporting probes combined with machine learning algorithms, as a general strategy for highly multiplexed biofluid assay.     

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers

Rapid and accurate diagnosis of COVID-19 plays an essential role in the current epidemic prevention and control. Despite the promise of nucleic acid and antibody tests, there is still a great challenge to reduce the misdiagnosis, especially for asymptomatic individuals. Here we report a generalizable method for highly specific and ultrasensitive detection of serum COVID-19-associated antigens based on an aptamer-assisted proximity ligation assay. The sensor is based on binding two aptamer probes to the same protein target that brings the ligation DNA region into close proximity, thereby initiating ligation-dependent qPCR amplification. Using this system, serum nucleocapsid protein has been detected quantitatively by converting protein recognition into a detectable qPCR signal using a simple, homogeneous and fast detection workflow in ∼2 hours. In addition, this system has also been transformed into a universal platform for measuring specific interactions between spike S1 and its receptor ACE2, and more importantly demonstrated the feasibility for screening and investigation of potential neutralizing aptamers. Since in vitro selection can obtain aptamers selective for many COVID-19-associated antigens, the method demonstrated here will serve as an important tool for the diagnosis and therapeutics of COVID-19.

A versatile aptamer-assisted proximity ligation system improves diagnosis of COVID-19, and allows the evaluation of potential neutralizing aptamers.     

Synthesis and molecular docking study of novel COVID-19 inhibitors

In 2020, the world tried to combat the corona virus (COVID-19) pandemic. A proven treatment method specific to Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is still not found. In this study, seven new antiviral compounds were designed for COVID-19 treatment. The ability of these compounds to inhibit COVID-19’s RNA processing was calculated by the molecular docking study. It has been observed that the compounds can have high binding affinities especially against NSP12 (between -9.06 and -8.00 kcal/mol). The molecular dynamics simulation of NSP12-ZG 7 complex proved the stability of interaction. The synthesis of two most active molecules was performed by one-pot reaction and characterized by FT-IR, ¹H-NMR, ¹³C-NMR, and mass spectroscopy. The compounds presented with their synthesis are inhibitory core structures against SARS-CoV-2 infection.     

基于成簇的规则间隔短回文重复序列的严重急性呼吸综合征冠状病毒2检测的最新进展北大核心CSCD

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)导致的新冠肺炎(COVID-19)迅速蔓延全球,给全球公共卫生系统带来了挑战。由于逆转录-定量聚合酶链反应(RT-qPCR)和抗原测试的普遍适用性和灵敏度较差,并且具有不同突变的SARS-CoV-2变体持续的出现,给疫情防控带来了更大的挑战,因此,高灵敏度、无需设备并且能够区分SARS-CoV-2变体的诊断方法亟须发展。基于成簇的规则间隔短回文重复序列(CRISPR)的诊断对设备要求低,具有可编程性、灵敏性和易用性,已经发展出多种核酸检测工具用于传染病的诊断,其在临床上具有巨大的应用潜力。文章聚焦于近期发表的基于CRISPR实现SARS-CoV-2检测和变体区分的最新技术,总结其特点并对其发展进行了展望。     

CRISPR-Mediated Profiling of Viral RNA at Single-Nucleotide Resolution

Mass pathogen screening is critical to preventing the outbreaks and spread of infectious diseases. The large-scale epidemic of COVID-19 and the rapid mutation of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus have put forward new requirements for virus detection and identification techniques. Here, we report a CRISPR-based Amplification-free Viral RNA Electrical Detection platform (CAVRED) for the rapid detection and identification of SARS-CoV-2 variants. A series of CRISPR RNA assays were designed to amplify the CRISPR-Cas system‘s ability to discriminate between mutant and wild RNA genomes with a single-nucleotide difference. The identified viral RNA information was converted into readable electrical signals through field-effect transistor biosensors for the achievement of highly sensitive detection of single-base mutations. CAVRED can detect the SARS-CoV-2 virus genome as low as 1 cp μL⁻¹ within 20 mins without amplification, and this value is comparable to the detection limit of real-time quantitative polymerase chain reaction. Based on the excellent RNA mutation detection ability, an 8-in-1 CAVRED array was constructed and realized the rapid identification of 40 simulated throat swab samples of SARS-CoV-2 variants with a 95.0 % accuracy. The advantages of accuracy, sensitivity, and fast speed of CAVRED promise its application in rapid and large-scale epidemic screening.     

Artemisia Extracts and Artemisinin-Based Antimalarials for COVID-19 Management: Could These Be Effective Antivirals for COVID-19 Treatment?

As the world desperately searches for ways to treat the coronavirus disease 2019 (COVID-19) pandemic, a growing number of people are turning to herbal remedies. The Artemisia species, such as A. annua and A. afra, in particular, exhibit positive effects against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and COVID-19 related symptoms. A. annua is a source of artemisinin, which is active against malaria, and also exhibits potential for other diseases. This has increased interest in artemisinin’s potential for drug repurposing. Artemisinin-based combination therapies, so-called ACTs, have already been recognized as first-line treatments against malaria. Artemisia extract, as well as ACTs, have demonstrated inhibition of SARS-CoV-2. Artemisinin and its derivatives have also shown anti-inflammatory effects, including inhibition of interleukin-6 (IL-6) that plays a key role in the development of severe COVID-19. There is now sufficient evidence in the literature to suggest the effectiveness of Artemisia, its constituents and/or artemisinin derivatives, to fight against the SARS-CoV-2 infection by inhibiting its invasion, and replication, as well as reducing oxidative stress and inflammation, and mitigating lung damage.     

Influence of particle size on the SARS-CoV-2 spike protein detection using IgG-capped gold nanoparticles and dynamic light scattering

Due to the unprecedented and ongoing nature of the coronavirus outbreak, the development of rapid immunoassays to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its highly contagious variants is an important and challenging task. Here, we report the development of polyclonal antibody-functionalized spherical gold nanoparticle biosensors as well as the influence of the nanoparticle sizes on the immunoassay response to detect the SARS-CoV-2 spike protein by dynamic light scattering. By monitoring the increment in the hydrodynamic diameter (ΔD_H) by dynamic light scattering measurements in the antigen–antibody interaction, SARS-CoV-2 S-protein can be detected in only 5 min. The larger the nanoparticles, the larger ΔD_H in the presence of spike protein. From adsorption isotherm, the calculated binding constant (K_D) was 83 nM and the estimated limit of detection was 13 ng/mL (30 pM). The biosensor was stable up to 90 days at 4 °C. Therefore, the biosensor developed in this work could be potentially applied as a fast and sensible immunoassay to detect SARS-CoV-2 infection in patient samples.     

In-silico investigation of phenolic compounds from leaves of Phillyrea angustifolia L. as a potential inhibitor against the SARS-CoV-2 main protease (Mpro PDB ID:5R83) using a virtual screening method

There is currently a global COVID-19 pandemic caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) and its variants. This highly contagious viral disease continues to pose a major health threat global. The discovery of vaccinations is not enough to prevent their spread and dire consequences. To take advantage of the current drugs and isolated compounds, and immediately qualifying approach is required. The aim of our research is evaluation the potency for natural antiviral compounds against the SARS CoV-2 M^pro. Molecular docking of four phenolic compounds from Phillyrea angustifolia leaves with SARS-CoV-2 M^pro has been conducted. Similarly, the stability of selected ligand–protein interactions has been determined using MD simulations. Moreover, the quantitative structure–activity relationship (QSAR), MMGBSA binding energies, pharmacokinetics, and drug-likeness predictions for selected phenolic have been reported. The selected phenolic compounds (Luteolin-7-O-glucoside, Apigenin-7-O-glucoside, Demethyl-oleuropein, and Oleuropein aglycone) revealed strong binding contacts in the two active pockets of a target protein of SARS-CoV-2 M^pro with the docking scores and highest binding energies with a binding energy of ?8.2 kcal/mol; ?7.8 kcal/mol; ?7.2 kcal/mol and ?7.0 kcal/mol respectively. Both Demethyloleoeuropein and Oleuropein aglycone can interact with residues His41 and Cys145 (catalytic dyad) and other amino acids of the binding pocket of M^pro. According to QSAR, studies on pharmacokinetics and drug-like properties suggested that oleuropein aglycone could be the best inhibitor of SARS-CoV-2 for new drug design and development. Further in vivo, in vitro, and clinical studies are highly needed to examine the potential of these phenolic compounds in the fight against COVID-19.     

Modelling the active SARS-CoV-2 helicase complex as a basis for structure-based inhibitor design

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19. We present here structural models and dynamics of the helicase in complex with its native substrates based on thorough analysis of homologous sequences and existing experimental structures. We performed and analysed microseconds of molecular dynamics (MD) simulations, and our model provides valuable insights to the binding of the ATP and ssRNA at the atomic level. We identify the principal motions characterising the enzyme and highlight the effect of the natural substrates on this dynamics. Furthermore, allosteric binding sites are suggested by our pocket analysis. Our obtained structural and dynamical insights are important for subsequent studies of the catalytic function and for the development of specific inhibitors at our characterised binding pockets for this promising COVID-19 drug target.

The RNA helicase (non-structural protein 13, NSP13) of SARS-CoV-2 is essential for viral replication, and it is highly conserved among the coronaviridae family, thus a prominent drug target to treat COVID-19.     

Chemiluminescent Imaging Assay of SARS-CoV-2 Protein with Target-Induced Enzyme Activity Regulation

Simple but robust testing assays are essential for screening and diagnosis of individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in COVID-19 pandemic. Here, we described a chemiluminescent imaging assay (CLIA) for sensitive and convenient detection of SARS-CoV-2 nucleocapsid protein (NP) by a target-induced enzyme activity regulation (T-EAR) strategy. The T-EAR used a pair of antibody-DNA probes to recognize SARS-CoV-2 NP and proximity-induce rolling circle amplification for mass-production of pyrophosphate to coordinate with Cu²⁺, which prevented the reduction of Cu²⁺ to Cu⁺ by sodium ascorbate as well as the Cu⁺-caused inactivation of horseradish peroxidase (HRP). The activity retention of HRP produced strong CL signal for the detection of SARS-CoV-2 NP by catalyzing the oxidation of luminol by H₂O₂. The T-EAR based CLIA showed a wide detection range from 1 pg/mL to 100 ng/mL (13 fM to 1.3 nM) with the requirement of only 0.75 μL of sample. This CLIA had advantages of good sensitivity, simple wash-free operation, acceptable accuracy, and high-throughput imaging detection, displaying potential applicability in screening assay of COVID-19 infection.     

Platycodin D,a natural component of Platycodon grandiflorum,prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann–Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.Subject terms: Viral infection, Lipid signalling     

Development of polypyrrole (nano)structures decorated with gold nanoparticles toward immunosensing for COVID-19 serological diagnosis

The rapid and reliable detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) seroconversion in humans is crucial for suitable infection control. In this sense, many studies have focused on increasing the sensibility, lowering the detection limits and minimizing false negative/positive results. Thus, biosensors based on nanoarchitectures of conducting polymers are promising alternatives to more traditional materials since they can hold improved surface area, higher electrical conductivity and electrochemical activity. In this work, we reported the analytical comparison of two different conducting polymers morphologies for the development of an impedimetric biosensor to monitor SARS-CoV-2 seroconversion in humans. Biosensors based on polypyrrole (PPy), synthesized in both globular and nanotubular (NT) morphology, and gold nanoparticles are reported, using a self-assembly monolayer of 3-mercaptopropionic acid and covalently linked SARS-CoV-2 Nucleocapsid protein. First, the novel hybrid materials were characterized by electron microscopy and electrochemical measurements, and the biosensor step-by-step construction was characterized by electrochemical and spectroscopic techniques. As a proof of concept, the biosensor was used for the impedimetric detection of anti-SARS-CoV-2 Nucleocapsid protein monoclonal antibodies. The results showed a linear response for different antibody concentrations, good sensibility and possibility to quantify 7.442 and 0.4 ng/mL of monoclonal antibody for PPy in the globular and NT morphology, respectively. The PPy-NTs biosensor was able to discriminate serum obtained from COVID-19 positive versus negative clinical samples and is a promising tool for COVID-19 immunodiagnostic, which can contribute to further studies concerning rapid, efficient, and reliable detections.     

In silico studies on phytochemicals to combat the emerging COVID-19 infection

The current COVID-19 pandemic, caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its variants, remains a serious health hazard globally. The SARS-CoV-2 Mpro and spike proteins, as well as the human ACE2 receptor, have previously been reported as good targets for the development of new drug leads to combat COVID-19. Various ligands, including synthetic and plant-derived small molecules, can interact with the aforementioned proteins. In this study, we investigated the interaction of eight phytochemicals, from selected medicinal plants (Aegle marmelos, Azadirachta indica, and Ocimum sanctum) commonly used in Indian traditional medicine, with SARS-CoV-2 Mpro (PDBID: 6LU7), SARS-CoV-2S spike protein (PDB ID: 6M0J) and the human ACE2 receptor (PDB ID: 6M18). All compounds were subjected to density functional theory (DFT) and frontier molecular orbitals (FMO) analysis to determine their geometry, and key electronic and energetic properties. Upon examining the interactions of the phytochemicals with the human ACE2 receptor and the SARS-CoV-2 Mpro, spike protein targets, two compounds (C-5 and C-8) were identified as the best binding ligands. These were further examined in MD simulation studies to determine the stability of the ligand–protein interactions. QSAR, pharmacokinetic and drug-likeness properties studies revealed that C-5 may be the best candidate to serve as a template for the design and development of new drugs to combat COVID-19.     

A Comprehensive Review of Andrographis paniculata (Burm. f.) Nees and Its Constituents as Potential Lead Compounds for COVID-19 Drug Discovery

The COVID-19 pandemic has intensively disrupted global health, economics, and well-being. Andrographis paniculata (Burm. f.) Nees has been used as a complementary treatment for COVID-19 in several Asian countries. This review aimed to summarize the information available regarding A. paniculata and its constituents, to provide critical points relating to its pharmacological properties, safety, and efficacy, revealing its potential to serve as a source of lead compounds for COVID-19 drug discovery. A. paniculata and its active compounds possess favorable antiviral, anti-inflammatory, immunomodulatory, and antipyretic activities that could be beneficial for COVID-19 treatment. Interestingly, recent in silico and in vitro studies have revealed that the active ingredients in A. paniculata showed promising activities against 3CLpro and its virus-specific target protein, human hACE2 protein; they also inhibit infectious virion production. Moreover, existing publications regarding randomized controlled trials demonstrated that the use of A. paniculata alone or in combination was superior to the placebo in reducing the severity of upper respiratory tract infection (URTI) manifestations, especially as part of early treatment, without serious side effects. Taken together, its chemical and biological properties, especially its antiviral activities against SARS-CoV-2, clinical trials on URTI, and the safety of A. paniculata, as discussed in this review, support the argument that A. paniculata is a promising natural source for drug discovery regarding COVID-19 post-infectious treatment, rather than prophylaxis.