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

Abstract

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.

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

“…The first step consisted of purifying highly sensitive antibodies from human samples through standard affinity-based methods. The produced antibodies were carefully characterized by various techniques such as ELISA, SDS-PAGE, LC-Q-TOF–MS/MS, and electrochemical analysis to validate the purity and functionality of the proteins [ 3 , 18 ]. The purified antibodies were conjugated with the synthesized PPy- g -PCL using succinic anhydride and EDC/NHS chemistries.…”

“…Their application covers a wide range of targets, including viral detection, substance use, toxins, environmental monitoring, food safety, and many others [ 2 ]. Immunoassays are qualitative and quantitative tools for detecting and measuring various targets based on the interaction between an antibody and a target antigen, making them a highly sensitive and selective instrument for diagnosis [ 3 ]. Immunoassay approaches have been integrated into many POC tests, such as paper-based assays (lateral flow assays and dot blots).…”

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

“…Additionally, it helps predict immune response patterns that are necessary for developing prevention Placeholder TextPeptides with 7 amino acids or higher are considered as plausible sequences for further analyses methods. A more thorough study of the application of the current purified antigens and antibodies has been reported by our research group in our recent publication [13].…”

“…The color signal was provided by synthesizing a dyeloaded polymersome that was bioconjugated with the purified antibody cocktail and loaded with Coomassie blue dye. The detailed protocol for the biosensor's design, preparation, and application can be found in our recent work [13].…”

“…The purified antibodies show great potential as a tool to be explored in diagnostic procedures such as ELISA, lateral flow assay, biosensors, etc. We have explored the potential of the purified antigens and antibodies in the current study by developing a colorimetric paper-based LFA where the system efficiently recognized positive and negative samples with a sensitivity of 93% compared with RT-PCR results [13]. These materials can also be exploited in combination with mass spectrometry to explore the antigen sequences for identification.…”