Monkeypox, a rare zoonotic disease classified as a category B infectious disease, is caused by the monkeypox virus (MPXV), which has a broad host range. Since May 2022, human MPXV infection outbreak has spread to more than 100 countries, and China has reported 106 MPXV infections between June 2 and 30, 2023. Therefore, rapid and effective MPXV detection is essential to control and prevent its transmission. In this study, a rapid, cost-effective, and convenient fluorescent immunochromatographic assay (FICA) for MPXV antigen A35R detection was developed. Under the optimal reaction conditions, the visual detection limit of the proposed assay was 7.5 ng/mL and was completed within 15 min. There was no cross-reactivity with norovirus (NV) or dengue virus (DENV) controls. Additionally, the clinical application of the proposed method was demonstrated by testing human serum samples spiked with the MPXV antigen A35R. FICA was faster and more convenient than traditional enzyme-linked immunosorbent assays (ELISA) or polymerase chain reaction (PCR), and hence suitable for the early screening of MPXV infection. In summary, FICA could realize rapid, low-cost, sensitive, and specific MPXV detection hold potential application prospects in clinical settings.
Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have been considered as the next generation molecular diagnosis tool. Single-readout mode has been extensively employed in massive CRISPR/Cas12a-based biosensors. In this work, we propose a one-tube dual-readout biosensor for ultrasensitive nucleic acids and non-nucleic acids detection by harnessing CRISPR-ALP tandem assay for the first time (termed CRISAT). In the presence of target, the Cas12a is activated to randomly cut the single-stranded hyDNA sequence of MB@hyDNA-cALP, thus releasing abundant alkaline phosphatase (ALP) in the supernatant solution. By using 4-aminophenol phosphate as the substrate of ALP, p-aminophenol is produced and then reacts with N-[3-(trimethoxysilyl)propyl]ethylenediamine or diethylenetriamine to generate silicon-containing polymer carbon dots (Si PCDs) or polymer carbon dots (PCDs) in situ, which can be observed by naked eye or detected by a fluorescent device in the same solution. Using this strategy, a fluoroscence and colorimetry dual-readout nanoplatform for CRISPR-based biosensor can be rationally developed. We ascertain the applicability of CRISAT through detecting SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can be also employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work provides a great potential for nucleic acids and non-nucleic acids detection.
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