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We report the development of a label-, antibody-, enzyme-, and amplification-free ratiometric fluorescent biosensor for low-cost and rapid (less than 12 min) diagnosis of COVID-19 from isolated RNA samples. The biosensor is designed on the basis of cytosine-modified antisense oligonucleotides specific for either N gene or RdRP gene that can form silver nanoclusters (AgNCs) with both green and red emission on an oligonucleotide via a one-step synthesis process. The presence of the target RNA sequence of SARS-CoV-2 causes a dual-emission ratiometric signal transduction, resulting in a limit of detection of 0.30 to 10.0 nM and appropriate linear ranges with no need for any further amplification, fluorophore, or design with a special DNA fragment. With this strategy, five different ratiometric fluorescent probes are designed, and how the T/C ratio, the length of the stem region, and the number of cytosines in the loop structure and at the 3′ end of the cluster-stabilizing template can affect the biosensor sensitivity is investigated. Furthermore, the effect of graphene oxide (GO) on the ratiometric behavior of nanoclusters is demonstrated and the concentration-/time-dependent new competitive mechanism between aggregation-caused quenching (ACQ) and aggregation-induced emission enhancement (AIE) for the developed ssDNA-AgNCs/GO nanohybrids is proposed. Finally, the performance of the designed ratiometric biosensor has been validated using the RNA extract obtained from more than 150 clinical samples, and the results have been confirmed by the FDA-approved reverse transcription-polymerase chain reaction (RT-PCR) diagnostic method. The diagnostic sensitivity and specificity of the best probe is more than >90%, with an area under the receiver operating characteristic (ROC) curve of 0.978.

Background: Pasteurella multocida is the causative agent of many diseases. Antimicrobial treatment disadvantages highlight the need to find other possible ways such as prophylaxis to manage infections. Current vaccines against this agent include inactivated bacteria, live-attenuated bacteria, and nonpathogenic bacteria, which have disadvantages such as lack of immunogenicity, reactogenicity, or reversion to virulence wild bacteria. Using bioinformatical approaches, potentially immunogenic and protective epitopes identified and merged to design the best epitope fusion form in case of immunogenicity as a vaccine candidate. Materials and Methods: In this study, the fusion protein ( PlpE1 + 2 + 3 ) and full PlpE genes ( PlpE-Total ) were cloned in pET28a in BL21 (DE3) firstly and later in pBAD/gIII A and expressed in Top10 Escherichia coli. Overlap polymerase chain reaction (PCR) using different primers for 5ˈ and 3ˈ end of each segment produced fusion segment 1 + 2 and (1 + 2) +3 fragments and was used for cloning. Results: Cloning of both PlpE1 + 2 + 3 and PlpE-Total into the pET28a vector and their transform into the BL21 (DE3) E. coli host was successful, as the presence of the cassettes was proved by digestion and colony PCR, however, their expression faced some challenges independent of expression inducer (isopropyl β-d-1-thiogalactopyranoside) concentration. Conclusion: Changing the vector to pBAD/gIII A and consequently changing the host to Top10 E. coli have resulted in sufficient expression, which shows that Top10 E. coli may be a good substitute for such cases. Furthermore, it is concluded that adding 8M urea results in sufficient purification, which hypothesizes that denature purification is better for such cases than native one. Purified proteins headed for further analysis as vaccine candidates.