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.
Carbon nanotubes (CNTs) are allotropes of carbon, which have unique physical, mechanical, and electronic properties. Among various biomedical applications, CNTs also attract interest as nonviral gene delivery systems. Functionalization of CNTs with cationic groups enables delivery of negatively charged DNA into cells. In contrast to this well-known strategy for DNA delivery, our approach included the covalent attachment of linearized plasmid DNA to carboxylated multiwalled CNTs (MWCNTs). Carboxyl groups were introduced onto MWCNTs by oxidative treatment, and then the carboxyl groups were activated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The whole pQE-70 vector including the gene encoding green fluorescent protein (GFP) was subjected to polymerase chain reaction (PCR) using the modified nucleotide N6-(6-Amino)hexyl-2'-deoxyadenosine-5'-triphosphate. Hence, free amino groups were introduced onto the linearized plasmid. Covalent bonding between the amino-modified plasmid DNA and the carboxylated MWCNTs was achieved via EDC chemistry. The resulting bioconjugate was successfully transformed into chemically competent Escherichia coli cells, without necessity of a heat-shock step at 42°C. The presence of Ca(2+) in transformation medium was required to neutralize the electrostatic repulsion between DNA and negatively charged outer layer of E. coli. The transformants, which were able to express GFP were inspected manually on ampicillin agar plates. Our study represents a novelty with respect to other noncovalent CNT gene delivery systems. Considering the interest for delivery of linear DNA fragments, our study could give insights into further studies.
Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (βα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.
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