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Temperature responsive smart polymer for enabling affinity enrichment of current coronavirus (SARS-CoV-2) to improve its diagnostic sensitivity.

ABSTRACT: The current commercially available SARS-CoV-2 diagnostic approaches including nucleic acid molecular assaying using polymerase chain reaction (PCR) have many limitations and drawbacks. SARS-CoV-2 diagnostic strategies were reported to have a high false-negative rate and low sensitivity due to low viral antibodies or antigenic load in the specimens, that is why even PCR test is recommended to be repeated to overcome this problem. Thus, in anticipation of COVID-19 current wave and the upcoming waves, we should have an accurate and rapid diagnostic tool to control this pandemic. In this study, we developed a novel preanalytical strategy to be used for SARS-CoV-2 specimen enrichment to avoid misdiagnosis. This method depends on the immuno-affinity trapping of the viral target followed by in situ thermal precipitation and enrichment. We designed, synthesized, and characterized a thermal-responsive polymer poly (N-isopropylacrylamide-co-2-hydroxyisopropylacrylamide-co-strained alkyne isopropylacrylamide) followed by decoration with SARS-CoV-2 antibody. Different investigations approved the successful synthesis of the polymeric antibody conjugate. This conjugate was shown to enrich recombinant SARS-CoV-2 nucleocapsid protein samples to about 6 folds. This developed system succeeded in avoiding the misdiagnosis of low viral load specimens using the lateral flow immunoassay test. The strength of this work is that, to the best of our knowledge, this report may be the first to functionalize SARS-CoV-2 antibody to a thermo-responsive polymer for increasing its screening sensitivity during the current pandemic.

SUBMITTER: Nabil A

PROVIDER: S-EPMC8200327 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Immunoassays have been translated into microfluidic device formats, but significant challenges relating to upstream sample processing still limit their applications. Here, stimuli-responsive polymer-antibody conjugates are utilized in a microfluidic immunoassay to enable rapid biomarker purification and enrichment as well as sensitive detection. The conjugates were constructed by covalently grafting poly(N-isopropylacrylamide) (PNIPAAm), a thermally responsive polymer, to the lysine residues of anti-prostate specific antigen (PSA) Immunoglobulin G (IgG) using carbodiimide chemistry via the polymer end-carboxylate. The antibody-PNIPAAm (capture) conjugates and antibody-alkaline phosphatase (detection) conjugates formed sandwich immunocomplexes via PSA binding in 50% human plasma. The complexes were loaded into a recirculating poly(dimethylsiloxane) microreactor, equipped with micropumps and transverse flow features, for subsequent separation, enrichment, and quantification. The immunocomplexes were captured by heating the solution to 39 °C, mixed over the transverse features for 2 min, and washed with warm buffer. In one approach, the assay utilized immunocomplex solution that was contained in an 80 nL microreactor, which was loaded with solution at room temperature and subsequently heated to 39 °C. The assay took 25 min and resulted in 37 pM PSA limit of detection (LOD), which is comparable to a plate ELISA employing the same antibody pair. In another approach, the microreactor was preheated to 39 °C, and immunocomplex solution was flowed through the reactor, mixed, and washed. When the specimen volume was increased to 7.5 μL by repeating the capture process three times, the higher specimen volume led to immunocomplex enrichment within the microreactor. The resulting assay LOD was 0.5 pM, which is 2 orders of magnitude lower than the plate ELISA. Both approaches generate antigen specific signal over a clinically significant range. The sample processing capabilities and subsequent utility in a biomarker assay demonstrate the opportunity for stimuli-responsive polymer-protein conjugates in novel diagnostic technologies.

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Temperature responsive smart polymer for enabling affinity enrichment of current coronavirus (SARS-CoV-2) to improve its diagnostic sensitivity.

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