A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5′ end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3′ end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5′-GGATCC-3′ between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas® Amplicor HCV Test Analyzer.
Digital detection strategy Hepatitis C virus RNA Glucose-loaded liposome Personal glucometer BamHI endonuclease
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This work was financially supported by the National Natural Science Foundation of China (grant no.: 81703477), and the Earmarked Funds Provided by the Science and Technology Plan Projects of Putian City (grant no.: 2018).
Compliance with ethical standards
All procedures performed in studies involving human participants were approved by the Affiliated Hospital of Putian University and were carried out in accordance with the ethical standards of the Fujian Provincial Research Committee and the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.
Informed consent was obtained individually from all participants included in this study.
Conflict of interest
The authors declare that they have no competing interests.
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