'All In One' SARS-CoV-2 variant recognition platform: Machine learning-enabled point of care diagnostics.

Point of care (PoC) devices are highly demanding to control current pandemic, originated from severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Though nucleic acid-based methods such as RT-PCR are widely available, they require sample preparation and long processing time. PoC diagnostic devices provide relatively faster and stable results. However they require further investigation to provide high accuracy and be adaptable for the new variants. In this study, laser-scribed graphene (LSG) sensors are coupled with gold nanoparticles (AuNPs) as stable promising biosensing platforms. Angiotensin Converting Enzyme 2 (ACE2), an enzymatic receptor, is chosen to be the biorecognition unit due to its high binding affinity towards spike proteins as a key-lock model. The sensor was integrated to a homemade and portable potentistat device, wirelessly connected to a smartphone having a customized application for easy operation. LODs of 5.14 and 2.09 ng/mL was achieved for S1 and S2 protein in the linear range of 1.0-200 ng/mL, respectively. Clinical study has been conducted with nasopharyngeal swabs from 63 patients having alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2) variants, patients without mutation and negative patients. A machine learning model was developed with accuracy of 99.37% for the identification of the SARS-Cov-2 variants under 1 min. With the increasing need for rapid and improved disease diagnosis and monitoring, the PoC platform proved its potential for real time monitoring by providing accurate and fast variant identification without any expertise and pre sample preparation, which is exactly what societies need in this time of pandemic.

Fluorescent bioassay for SARS-CoV-2 detection using polypyrene-g-poly(ε-caprolactone) prepared by simultaneous photoinduced step-growth and ring-opening polymerizations.

The construction of a rapid and easy immunofluorescence bioassay for SARS-CoV-2 detection is described. We report for the first time a novel one-pot synthetic approach for simultaneous photoinduced step-growth polymerization of pyrene (Py) and ring-opening polymerization of ε-caprolactone (PCL) to produce a graft fluorescent copolymer PPy-g-PCL that was conjugated to SARS-CoV-2-specific antibodies using EDC/NHS chemistry. The synthesis steps and conjugation products were fully characterized using standard spectral analysis. Next, the PPy-g-PCL was used for the construction of a dot-blot assay which was calibrated for applications to human nasopharyngeal samples. The analytical features of the proposed sensor showed a detection range of 6.03-8.7 LOG viral copy mL-1 (Ct Scores: 8-25), the limit of detection (LOD), and quantification (LOQ) of 1.84 and 6.16 LOG viral copy mL-1, respectively. The repeatability and reproducibility of the platform had a coefficient of variation (CV) ranging between 1.2 and 5.9%. The fluorescence-based dot-blot assay was tested with human samples. Significant differences were observed between the fluorescence intensity of the negative and positive samples, with an overall correct response of 93.33%. The assay demonstrated a high correlation with RT-PCR data. This strategy opens new insights into simplified synthesis procedures of the reporter molecules and their high potential sensing and diagnosis applications.

Indiscriminate SARS-CoV-2 multivariant detection using magnetic nanoparticle-based electrochemical immunosensing.

The increasing mutation frequency of the SARS-CoV-2 virus and the emergence of successive variants have made correct diagnosis hard to perform. Developing efficient and accurate methods to diagnose infected patients is crucial to effectively mitigate the pandemic. Here, we developed an electrochemical immunosensor based on SARS-CoV-2 antibody cocktail-conjugated magnetic nanoparticles for the sensitive and accurate detection of the SARS-CoV-2 virus and its variants in nasopharyngeal swabs. The application of the antibody cocktail was compared with commercially available anti-SARS-CoV-2 S1 (anti-S1) and anti-S2 monoclonal antibodies. After optimization and calibration, the limit of detection (LOD) determination demonstrated a LOD = 0.53-0.75 ng/mL for the antibody cocktail-based sensor compared with 0.93 ng/mL and 0.99 ng/mL for the platforms using anti-S1 and anti-S2, respectively. The platforms were tested with human nasopharyngeal swab samples pre-diagnosed with RT-PCR (10 negatives and 40 positive samples). The positive samples include the original, alpha, beta, and delta variants (n = 10, for each). The polyclonal antibody cocktail performed better than commercial anti-S1 and anti-S2 antibodies for all samples reaching 100% overall sensitivity, specificity, and accuracy. It also showed a wide range of variants detection compared to monoclonal antibody-based platforms. The present work proposes a versatile electrochemical biosensor for the indiscriminate detection of the different variants of SARS-CoV-2 using a polyclonal antibody cocktail. Such diagnostic tools allowing the detection of variants can be of great efficiency and economic value in the fight against the ever-changing SARS-CoV-2 virus.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

Dye-Loaded Polymersome-Based Lateral Flow Assay: Rational Design of a COVID-19 Testing Platform by Repurposing SARS-CoV-2 Antibody Cocktail and Antigens Obtained from Positive Human Samples.

The global pandemic of COVID-19 continues to be an important threat, especially with the fast transmission rate observed after the discovery of novel mutations. In this perspective, prompt diagnosis requires massive economical and human resources to mitigate the disease. The current study proposes a rational design of a colorimetric lateral flow immunoassay (LFA) based on the repurposing of human samples to produce COVID-19-specific antigens and antibodies in combination with a novel dye-loaded polymersome for naked-eye detection. A group of 121 human samples (61 serums and 60 nasal swabs) were obtained and analyzed by RT-PCR and ELISA. Pooled samples were used to purify antibodies using affinity chromatography, while antigens were purified via magnetic nanoparticles-based affinity. The purified proteins were confirmed for their specificity to COVID-19 via commercial LFA, ELISA, and electrochemical tests in addition to sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Polymersomes were prepared using methoxy polyethylene glycol-b-polycaprolactone (mPEG-b-PCL) diblock copolymers and loaded with a Coomassie Blue dye. The polymersomes were then functionalized with the purified antibodies and applied for the preparation of two types of LFA (antigen test and antibody test). Overall, the proposed diagnostic tests demonstrated 93 and 92.2% sensitivity for antigen and antibody tests, respectively. The repeatability (92-94%) and reproducibility (96-98%) of the tests highlight the potential of the proposed LFA. The LFA test was also analyzed for stability, and after 4 weeks, 91-97% correct diagnosis was observed. The current LFA platform is a valuable assay that has great economical and analytical potential for widespread applications.

Quantitative paper-based dot blot assay for spike protein detection using fuchsine dye-loaded polymersomes.

Real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-based assays are the gold standard for virus diagnosis. Point-of-care (POC) technologies have shown great progress during this period. Herein, we propose a novel fuchsine dye-loaded polymersome for a colorimetric paper-based dot blot spike protein diagnostic assay for COVID-19 via smartphone-assisted sensing. The prepared platform aimed to create an adaptable tool that competes with traditional nanoparticle-based assays employing gold and silver. Analytical characterization and application of the testing platform showed high sensitivity (10 times better than gold nanoparticles), stability, fast turnaround, and reproducibility. The potential and possibilities demonstrated by the current platform could be observed in its adaptability for different markers and pathologies. In addition, smartphone-assisted sensing emphasizes the ability to use the tool at home by common peoples which can lower the burden on the healthcare facilities and reach more underdeveloped regions.

Rapid Point-of-Care COVID-19 Diagnosis with a Gold-Nanoarchitecture-Assisted Laser-Scribed Graphene Biosensor.

The global pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has revealed the urgent need for accurate, rapid, and affordable diagnostic tests for epidemic understanding and management by monitoring the population worldwide. Though current diagnostic methods including real-time polymerase chain reaction (RT-PCR) provide sensitive detection of SARS-CoV-2, they require relatively long processing time, equipped laboratory facilities, and highly skilled personnel. Laser-scribed graphene (LSG)-based biosensing platforms have gained enormous attention as miniaturized electrochemical systems, holding an enormous potential as point-of-care (POC) diagnostic tools. We describe here a miniaturized LSG-based electrochemical sensing scheme for coronavirus disease 2019 (COVID-19) diagnosis combined with three-dimensional (3D) gold nanostructures. This electrode was modified with the SARS-CoV-2 spike protein antibody following the proper surface modifications proved by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) characterizations as well as electrochemical techniques. The system was integrated into a handheld POC detection system operated using a custom smartphone application, providing a user-friendly diagnostic platform due to its ease of operation, accessibility, and systematic data management. The analytical features of the electrochemical immunoassay were evaluated using the standard solution of S-protein in the range of 5.0-500 ng/mL with a detection limit of 2.9 ng/mL. A clinical study was carried out on 23 patient blood serum samples with successful COVID-19 diagnosis, compared to the commercial RT-PCR, antibody blood test, and enzyme-linked immunosorbent assay (ELISA) IgG and IgA test results. Our test provides faster results compared to commercial diagnostic tools and offers a promising alternative solution for next-generation POC applications.

COVID-19 Pandemic and the Global Perspective of Turkish Thoracic Society.

It has been more than 3 months now since the first case of COVID-19 was reported in Turkey. Globally, the number of confirmed cases and deaths reached 9,653,048 and 491,128 respectively, as reported by 216 countries by June 27, 2020. Turkey had 1,396 new cases, 194,511 total cases, and 5,065 deaths by the same date. From the first case until today, the Turkish Thoracic Society (TTS) has been very proactive in educating doctors, increasing public awareness, undertaking academic studies, and assisting with public health policies. In the present report, social, academic, and management perspectives of the pandemic are presented under appropriate subtitles. During this critical public health crisis, TTS has once again demonstrated its readiness and constructive stance by supporting public health, healthcare workers, and the environment. This review summarizes the perspective of TTS on each aspect of the COVID-19 pandemic and casts light on its contributions.