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Abstract The COVID-19 pandemic has profoundly impacted global economies and healthcare systems, revealing critical vulnerabilities in both. In response, our study introduces a sensitive and highly specific detection method for cDNA, leveraging Luminescence Resonance Energy Transfer (LRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs), and achieves a detection limit of 242 fM for SARS-CoV-2 cDNA. This innovative sensing platform utilizes UCNPs conjugated with one primer and AuNPs with another, targeting the 5′ and 3′ ends of the SARS-CoV-2 cDNA, respectively, enabling precise differentiation of mismatched cDNA sequences and significantly improving detection specificity. Through rigorous experimental analysis, we established a quenching efficiency range from 10.4 % to 73.6 %, with an optimal midpoint of 42 %, thereby demonstrating the superior sensitivity of our method. Our work uses SARS-CoV-2 cDNA as a model system to demonstrate the potential of our LRET-based detection method. This proof-of-concept study highlights the adaptability of our platform for future diagnostic applications. Instrumental validation confirms the synthesis and formation of AuNPs, addressing the need for experimental verification of the preparation of nanomaterial. Our comparative analysis with existing SARS-CoV-2 detection methods revealed that our approach provides a low detection limit and high specificity for target cDNA sequences, underscoring its potential for targeted COVID-19 diagnostics. This study demonstrates the superior sensitivity and adaptability of using UCNPs and AuNPs for cDNA detection, offering significant advances in rapid, accessible diagnostic technologies. Our method, characterized by its low detection limit and high precision, represents a critical step forward in developing next-generation biosensors for managing current and future viral outbreaks. By adjusting primer sequences, this platform can be tailored to detect other pathogens, contributing to the enhancement of global healthcare responsiveness and infectious disease control. 

Abstract In a viral pandemic, a few important tests are required for successful containment of the virus and reduction in severity of the infection. Among those tests, a test for the neutralizing ability of an antibody is crucial for assessment of population immunity gained through vaccination, and to test therapeutic value of antibodies made to counter the infections. Here, we report a sensitive technique to detect the relative neutralizing strength of various antibodies against the SARS-CoV-2 virus. We used bright, photostable, background-free, fluorescent upconversion nanoparticles conjugated with SARS-CoV-2 receptor binding domain as a phantom virion. A glass bottom plate coated with angiotensin-converting enzyme 2 (ACE-2) protein imitates the target cells. When no neutralizing IgG antibody was present in the sample, the particles would bind to the ACE-2 with high affinity. In contrast, a neutralizing antibody can prevent particle attachment to the ACE-2-coated substrate. A prototype system consisting of a custom-made confocal microscope was used to quantify particle attachment to the substrate. The sensitivity of this assay can reach 4.0 ng/ml and the dynamic range is from 1.0 ng/ml to 3.2  $$\upmu$$ μ g/ml. This is to be compared to 19 ng/ml sensitivity of commercially available kits.