Gold nanoparticle conjugate-based lateral flow immunoassay (LFIA) for rapid detection of RBD antigen of SARS-CoV-2 in clinical samples using a smartphone-based application.

The coronavirus disease 2019 (COVID-19) pandemic has emphasized the need for development of a rapid diagnostic device for the effective treatment and its mitigation. Lateral flow immunoassay (LFIA) belongs to a class of diagnostic devices, which has the benefit of providing quick results, easy to handle, low cost, and on-site applicable. So far, several LFIA has been developed for the detection of infectious severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), however, only a few of them are antigen (Ag)-based. Here, we describe an antibody (Ab)-labeled gold nanoparticles (AuNPs)-based LFIA (AuNPs-LFIA) for the detection of Receptor-Binding Domain (RBD) of SARS-CoV-2. For this, RBD Ab of SARS-CoV-2 was conjugated with the AuNPs, which served as a detecting probe. The fabricated LFIA strip was optimized for different parameters such as membrane pore size, blocking conditions, Ab coating concentration, and conjugate incubation. The optimized LFIA strips were validated in spiked buffer samples and the optimal limit of detection was found to be 1 ng/ml, which was confirmed by a smartphone-based application. Moreover, the developed AuNPs-LFIA strips effectively detected RBD Ag in 100 clinical samples with 94.3% sensitivity and 90.9% specificity in clinical samples when compared with the gold standard (RT-PCR). The fabricated LFIAs are reported to have storage stability of up to 21 days at 4°C and room temperature (RT). Hence, the developed LFIA can be used as a portable, cost-effective diagnostic device for rapid detection of SARS-CoV-2.

Emerging 0D, 1D, 2D, and 3D nanostructures for efficient point-of-care biosensing.

The recent COVID-19 infection outbreak has raised the demand for rapid, highly sensitive POC biosensing technology for intelligent health and wellness. In this direction, efforts are being made to explore high-performance nano-systems for developing novel sensing technologies capable of functioning at point-of-care (POC) applications for quick diagnosis, data acquisition, and disease management. A combination of nanostructures [i.e., 0D (nanoparticles & quantum dots), 1D (nanorods, nanofibers, nanopillars, & nanowires), 2D (nanosheets, nanoplates, nanopores) & 3D nanomaterials (nanocomposites and complex hierarchical structures)], biosensing prototype, and micro-electronics makes biosensing suitable for early diagnosis, detection & prevention of life-threatening diseases. However, a knowledge gap associated with the potential of 0D, 1D, 2D, and 3D nanostructures for the design and development of efficient POC sensing is yet to be explored carefully and critically. With this focus, this review highlights the latest engineered 0D, 1D, 2D, and 3D nanomaterials for developing next-generation miniaturized, portable POC biosensors development to achieve high sensitivity with potential integration with the internet of medical things (IoMT, for miniaturization and data collection, security, and sharing), artificial intelligence (AI, for desired analytics), etc. for better diagnosis and disease management at the personalized level.

Graphene-Based Field-Effect Transistor for Ultrasensitive Immunosensing of SARS-CoV-2 Spike S1 Antigen.

Coronavirus disease (COVID-19) is an infectious disease that has posed a global health challenge caused by the SARS-CoV-2 virus. Early management and diagnosis of SARS-CoV-2 are crucial for the timely treatment, traceability, and reduction of viral spread. We have developed a rapid method using a Graphene-based Field-Effect Transistor (Gr-FET) for the ultrasensitive detection of SARS-CoV-2 Spike S1 antigen (S1-Ag). The in-house developed antispike S1 antibody (S1-Ab) was covalently immobilized on the surface of a carboxy functionalized graphene channel using carbodiimide chemistry. Ultraviolet-visible spectroscopy, Fourier-Transform Infrared Spectroscopy, X-ray Photoelectron Spectroscopy (XPS), Atomic Force Microscopy (AFM), Optical Microscopy, Raman Spectroscopy, Scanning Electron Microscopy (SEM), Enzyme-Linked Immunosorbent Assays (ELISA), and device stability studies were conducted to characterize the bioconjugation and fabrication process of Gr-FET. In addition, the electrical response of the device was evaluated by monitoring the change in resistance caused by Ag-Ab interaction in real time. For S1-Ag, our Gr-FET devices were tested in the range of 1 fM to 1 µM with a limit of detection of 10 fM in the standard buffer. The fabricated devices are highly sensitive, specific, and capable of detecting low levels of S1-Ag.

Label free detection of SARS CoV-2 Receptor Binding Domain (RBD) protein by fabrication of gold nanorods deposited on electrochemical immunosensor (GDEI).

Coronavirus Disease 2019 (COVID-19) pandemic has shown the need for early diagnosis to manage infectious disease outbreaks. Here, we report a label free electrochemical Fluorine-Doped Tin Oxide (FTO) Immunosensor coupled with gold nanorods (GNRs) as an electron carrier for ultrasensitive detection of the Receptor Binding Domain (RBD) of SARS CoV-2 Spike protein. The RBD gene was cloned, and expressed in-house with confirmed molecular weight of ∼31 kDa via Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF). RBD antibodies (Ab) were generated to be used as a bioreceptor for sensor fabrication, and characterized using SDS-PAGE, Western Blot, and Enzyme-Linked Immunosorbent Assay (ELISA). GNRs were fabricated on the electrode surface, followed by immobilization of RBD Ab. The conjugation steps were confirmed by UV-Vis Spectroscopy, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Cyclic Voltammetry (CV), and Differential Pulse Voltammetry (DPV). The fabricated electrode was further optimized for maximum efficiency and output. The detection limit of the developed electrode was determined as 0.73 fM for RBD antigen (Ag). Furthermore, the patient nasopharyngeal samples were collected in Viral Transport Media (VTM), and tested on the sensor surface that resulted in detection of SARS CoV-2 within 30 s, which was further validated via Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, the immunosensor showed good repeatability, storage stability, and minimal cross reactivity against Middle East Respiratory Syndrome (MERS) spike protein. Along with ease of fabrication, the electrodes show future miniaturization potential for extensive and rapid screening of populations for COVID-19.

A Recent Update on Advanced Molecular Diagnostic Techniques for COVID-19 Pandemic: An Overview.

Coronavirus disease 2019 (COVID-19), which started out as an outbreak of pneumonia, has now turned into a pandemic due to its rapid transmission. Besides developing a vaccine, rapid, accurate, and cost-effective diagnosis is essential for monitoring and combating the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its related variants on time with precision and accuracy. Currently, the gold standard for detection of SARS-CoV-2 is Reverse Transcription Polymerase Chain Reaction (RT-PCR), but it lacks accuracy, is time-consuming and cumbersome, and fails to detect multi-variant forms of the virus. Herein, we have summarized conventional diagnostic methods such as Chest-CT (Computed Tomography), RT-PCR, Loop Mediated Isothermal Amplification (LAMP), Reverse Transcription-LAMP (RT-LAMP), as well new modern diagnostics such as CRISPR-Cas-based assays, Surface Enhanced Raman Spectroscopy (SERS), Lateral Flow Assays (LFA), Graphene-Field Effect Transistor (GraFET), electrochemical sensors, immunosensors, antisense oligonucleotides (ASOs)-based assays, and microarrays for SARS-CoV-2 detection. This review will also provide an insight into an ongoing research and the possibility of developing more economical tools to tackle the COVID-19 pandemic.

Label-free detection of SARS-CoV-2 Spike S1 antigen triggered by electroactive gold nanoparticles on antibody coated fluorine-doped tin oxide (FTO) electrode.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, also known as 2019-nCov or COVID-19) outbreak has become a huge public health issue due to its rapid transmission making it a global pandemic. Here, we report fabricated fluorine doped tin oxide (FTO) electrodes/gold nanoparticles (AuNPs) complex coupled with in-house developed SARS-CoV-2 spike S1 antibody (SARS-CoV-2 Ab) to measure the response with Cyclic Voltammetry (CV) and Differential Pulse Voltammetry (DPV). The biophysical characterisation of FTO/AuNPs/SARS-CoV-2Ab was done via UV-Visible spectroscopy, Dynamic Light Scattering (DLS), and Fourier Transform Infrared Spectroscopy (FT-IR). The fabricated FTO/AuNPs/SARS-CoV-2Ab immunosensor was optimised for response time, antibody concentration, temperature, and pH. Under optimum conditions, the FTO/AuNPs/Ab based immunosensor displayed high sensitivity with limit of detection (LOD) up to 0.63 fM in standard buffer and 120 fM in spiked saliva samples for detection of SARS-CoV-2 spike S1 antigen (Ag) with negligible cross reactivity Middle East Respiratory Syndrome (MERS) spike protein. The proposed FTO/AuNPs/SARS-CoV-2Ab based biosensor proved to be stable for up to 4 weeks and can be used as an alternative non-invasive diagnostic tool for the rapid, specific and sensitive detection of SARS-CoV-2 Spike Ag traces in clinical samples.

From Nanosystems to a Biosensing Prototype for an Efficient Diagnostic: A Special Issue in Honor of Professor Bansi D. Malhotra.

It has been proven that rapid bioinformatics analysis according to patient health profiles, in addition to biomarker detection at a low level, is emerging as essential to design an analytical diagnostics system to manage health intelligently in a personalized manner. Such objectives need an optimized combination of a nano-enabled sensing prototype, artificial intelligence (AI)-supported predictive analysis, and Internet of Medical Things (IoMT)-based bioinformatics analysis. Such a developed system began with a prototype demonstration of efficient diseases diagnostics performance is the future diseases management approach. To explore these aspects, the Special Issue planned for the nano-and micro-technology section of MDPI's Biosensors journal will honor and acknowledge the contributions of Prof. B.D. Malhotra, Ph.D., FNA, FNASc has made in the field of biosensors.

Identification of Unique Peptides for SARS-CoV-2 Diagnostics and Vaccine Development by an In Silico Proteomics Approach.

Ongoing evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus strains is posing new COVID-19 diagnosis and treatment challenges. To help efforts to meet these challenges we examined data acquired from proteomic analyses of human SARS-CoV-2-infected cell lines and samples from COVID-19 patients. Initially, 129 unique peptides were identified, which were rigorously evaluated for repeats, disorders, polymorphisms, antigenicity, immunogenicity, toxicity, allergens, sequence similarity to human proteins, and contributions from other potential cross-reacting pathogenic species or the human saliva microbiome. We also screened SARS-CoV-2-infected NBHE and A549 cell lines for presence of antigenic peptides, and identified paratope peptides from crystal structures of SARS-CoV-2 antigen-antibody complexes. We then selected four antigen peptides for docking with known viral unbound T-cell receptor (TCR), class I and II peptide major histocompatibility complex (pMHC), and identified paratope sequences. We also tested the paratope binding affinity of SARS-CoV T- and B-cell peptides that had been previously experimentally validated. The resultant antigenic peptides have high potential for generating SARS-CoV-2-specific antibodies, and the paratope peptides can be directly used to develop a COVID-19 diagnostics assay. The presented genomics and proteomics-based in-silico approaches have apparent utility for identifying new diagnostic peptides that could be used to fight SARS-CoV-2.

Biochemical composition, transmission and diagnosis of SARS-CoV-2.

Coronavirus disease 2019 (COVID-19) is a life-threatening respiratory infection caused by severe acute respiratory syndrome virus (SARS-CoV-2), a novel human coronavirus. COVID-19 was declared a pandemic by World Health Organization in March 2020 for its continuous and rapid spread worldwide. Rapidly emerging COVID-19 epicenters and mutants of concerns have created mammoth chaos in healthcare sectors across the globe. With over 185 million infections and approximately 4 million deaths globally, COVID-19 continues its unchecked spread despite all mitigation measures. Until effective and affordable antiretroviral drugs are made available and the population at large is vaccinated, timely diagnosis of the infection and adoption of COVID-appropriate behavior remains major tool available to curtail the still escalating COVID-19 pandemic. This review provides an updated overview of various techniques of COVID-19 testing in human samples and also discusses, in brief, the biochemical composition and mode of transmission of the SARS-CoV-2. Technological advancement in various molecular, serological and immunological techniques including mainly the reverse-transcription polymerase chain reaction (RT-PCR), CRISPR, lateral flow assays (LFAs), and immunosensors are reviewed.

SARS-CoV-2 transcriptome analysis and molecular cataloguing of immunodominant epitopes for multi-epitope based vaccine design.

Genomics-led researches are engaged in tracing virus expression pattern, and induced immune responses in human to develop effective vaccine against COVID-19. In this study, targeted expression profiling and differential gene expression analysis of major histocompatibility complexes and innate immune system genes were performed through SARS-CoV-2 infected RNA-seq data of human cell line, and virus transcriptome was generated for T-and B-cell epitope prediction. Docking studies of epitopes with MHC and B-cell receptors were performed to identify potential T-and B-cell epitopes. Transcriptome analysis revealed the specific multiple allele expressions in cell line, genes for elicited induce immune response, and virus gene expression. Proposed T- and B-cell epitopes have high potential to elicit equivalent immune responses caused by SARS-CoV-2 infection which can be useful to provide links between elicited immune response and virus gene expression. This study will facilitate in vitro and in vivo vaccine related research studies in disease control.

Is the Collapse of the Respiratory Center in the Brain Responsible for Respiratory Breakdown in COVID-19 Patients?

Following the identification of severe acute respiratory syndrome coronavirus (SARS-CoV) in 2002 and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012, we are now again facing a global highly pathogenic novel coronavirus (SARS-CoV-2) epidemic. Although the lungs are one of the most critically affected organs, several other organs, including the brain may also get infected. Here, we have highlighted that SARS-CoV-2 might infect the central nervous system (CNS) through the olfactory bulb. From the olfactory bulb, SARS-CoV-2 may target the deeper parts of the brain including the thalamus and brainstem by trans-synaptic transfer described for many other viral diseases. Following this, the virus might infect the respiratory center of brain, which could be accountable for the respiratory breakdown of COVID-19 patients. Therefore, it is important to screen the COVID-19 patients for neurological symptoms as well as possibility of the collapse of the respiratory center in the brainstem should be investigated in depth.