Portable high-throughput multimodal immunoassay platform for rapid on-site COVID-19 diagnostics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a causal agent of Coronavirus Disease 2019 (COVID-19) has led to the global pandemic. Though the real-time reverse transcription polymerase chain reaction (RT-PCR) acting as a gold-standard method has been widely used for COVID-19 diagnostics, it can hardly support rapid on-site applications or monitor the stage of disease development as well as to identify the infection and immune status of rehabilitation patients. To suit rapid on-site COVID-19 diagnostics under various application scenarios with an all-in-one device and simple detection reagents, we propose a high-throughput multimodal immunoassay platform with fluorescent, colorimetric, and chemiluminescent immunoassays on the same portable device and a multimodal reporter probe using quantum dot (QD) microspheres modified with horseradish peroxidase (HRP) coupled with goat anti-human IgG. The recombinant nucleocapsid protein fixed on a 96-well plate works as the capture probe. In the condition with the target under detection, both reporter and capture probes can be bound by such target. When illuminated by excitation light, fluorescence signals from QD microspheres can be collected for target quantification often at a fast speed. Additionally, when pursuing simple detection without using any sensing devices, HRP-catalyzed TMB colorimetric immunoassay is employed; and when pursuing highly sensitive detection, HRP-catalyzed luminol chemiluminescent immunoassay is established. Verified by the anti-SARS-CoV-2 N humanized antibody, the sensitivities of colorimetric, fluorescent, and chemiluminescent immunoassays are respectively 20, 80, and 640 times more sensitive than that of the lateral flow colloidal gold immunoassay strip. Additionally, such a platform can simultaneously detect multiple samples at the same time thus supporting high-throughput sensing; and all these detecting operations can be implemented on-site within 50 min relying on field-operable processing and field-portable devices. Such a high-throughput multimodal immunoassay platform can provide a new all-in-one solution for rapid on-site diagnostics of COVID-19 for different detecting purposes.

Sensitivity and Specificity of Rapid SARS-CoV-2 Antigen Detection Using Different Sampling Methods: A Clinical Unicentral Study.

Rapid antigen detection of SARS-CoV-2 has been widely used. However, there is no consensus on the best sampling method. This study aimed to determine the level of agreement between SARS-CoV-2 fluorescent detection and a real-time reverse-transcriptase polymerase chain reaction (rRT-PCR), using different swab methods. Fifty COVID-19 and twenty-six healthy patients were confirmed via rRT-PCR, and each patient was sampled via four swab methods: oropharyngeal (O), nasal (N), spit saliva (S), and combined O/N/S swabs. Each swab was analyzed using an immunofluorescent Quidel system. The combined O/N/S swab provided the highest sensitivity (86%; Kappa = 0.8), followed by nasal (76%; Kappa = 0.68), whereas the saliva revealed the lowest sensitivity (66%; kappa = 0.57). Further, when considering positive detection in any of the O, N, and S samples, excellent agreements with rRT-PCR were achieved (Kappa = 0.91 and 0.97, respectively). Finally, among multiple factors, only patient age revealed a significant negative association with antigenic detection in the saliva. It is concluded that immunofluorescent detection of SARS-CoV-2 antigen is a reliable method for rapid diagnosis under circumstances where at least two swabs, one nasal and one oropharyngeal, are analyzed. Alternatively, a single combined O/N/S swab would improve the sensitivity in contrast to each site swabbed alone.

Horseradish peroxidase-triggered direct in situ fluorescent immunoassay platform for sensing cardiac troponin I and SARS-CoV-2 nucleocapsid protein in serum.

Direct in situ fluorescent enzyme-linked immunosorbent assay (ELISA) is rarely investigated and reported. Herein, a direct in situ high-performance HRP-labeled fluorescent immunoassay platform was constructed. The platform was developed based on a rapid in situ fluorogenic reaction between Polyethyleneimine (PEI) and p-Phenylenediamine (PPD) analogues to generate fluorescent copolymer nanoparticles (FCNPs). The formation mechanism of FCNPs was found to be the oxidation of •OH radicals, which was further proved by nitrogen protection and scavenger of •OH radicals. Meantime, the fluorescence wavelength of FCNPs could be adjusted from 471 to 512 nm by introducing various substitution groups into the PPD structure. Using cardiac troponin I (cTnI) and SARS-CoV-2 nucleocapsid protein (N-protein) as the model antigens, the proposed fluorescent ELISA exhibited a wide dynamic range of 5-180 ng/mL and a low limit of detection (LOD) of 0.19 ng/mL for cTnI, and dynamic range of 0-120 ng/mL and a LOD of 0.33 ng/mL for SARS-CoV-2 N protein, respectively. Noteworthy, the proposed method was successful applied to evaluate the cTnI and SARS-CoV-2 N protein levels in serum with satisfied results. Therefore, the proposed platform paved ways for developing novel fluorescence-based HRP-labeled ELISA technologies and broadening biomarker related clinical diagnostics.