Lanthanide-Complex-Enhanced Bioorthogonal Branched DNA Amplification
Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:96 |
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| Enthalten in: |
Analytical chemistry - 96(2024), 4 vom: 30. Jan., Seite 1556-1564 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhao, Fang [VerfasserIn] |
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| Links: |
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| Themen: |
63231-63-0 |
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| Anmerkungen: |
Date Completed 31.01.2024 Date Revised 20.02.2024 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1021/acs.analchem.3c04274 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM367047713 |
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| 520 | |a Fluorescence in situ hybridization (FISH) is a widely used technique for detecting intracellular nucleic acids. However, its effectiveness in detecting low-copy nucleic acids is limited due to its low fluorescence intensity and background autofluorescence. To address these challenges, we present here an approach of lanthanide-complex-enhanced bioorthogonal-branched DNA amplification (LEBODA) with high sensitivity for in situ nuclear acid detection in single cells. The approach capitalizes on two levels of signal amplification. First, it utilizes click chemistry to directly link a substantial number of bridge probes to target-recognizing probes, providing an initial boost in signal intensity. Second, it incorporates high-density lanthanide complexes into each bridge probe, enabling secondary amplifications. Compared to the traditional "double Z" probes used in the RNAscope method, LEBODA exhibits 4 times the single enhancement for RNA detection signal with the click chemistry approach. Using SARS-CoV-2 pseudovirus-infected HeLa cells, we demonstrate the superiority in the detection of viral-infected cells in rare populations as low as 20% infectious rate. More encouragingly, the LEBODA approach can be adapted for DNA-FISH and single-molecule RNA-FISH, as well as other hybridization-based signal amplification methods. This adaptability broadens the potential applications of LEBODA in the sensitive detection of biomolecules, indicating promising prospects for future research and practical use | ||
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| 700 | 1 | |a Wen, Shihui |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Le |e verfasserin |4 aut | |
| 700 | 1 | |a Jin, Dayong |e verfasserin |4 aut | |
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