posted on 2024-01-12, 10:29authored byFang Zhao, Yunpeng Guan, Fei Su, Zhongbo Du, Shihui Wen, Le Zhang, Dayong Jin
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.