Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Su, F; Chen, S; Liu, Y; Zhou, J; Du, Z; Luo, X; Wen, S; Jin, D

Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.

Su, F Chen, S Liu, Y Zhou, J

Du, Z Luo, X Wen, S

Jin, D

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Publisher:: AMER CHEMICAL SOC
Publication Type:: Journal Article
Citation:: Anal Chem, 2024, 96, (11), pp. 4430-4436
Issue Date:: 2024-03-19

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Field	Value	Language
dc.contributor.author	Su, F
dc.contributor.author	Chen, S
dc.contributor.author	Liu, Y
dc.contributor.author	Zhou, J https://orcid.org/0000-0002-0605-5745
dc.contributor.author	Du, Z
dc.contributor.author	Luo, X
dc.contributor.author	Wen, S https://orcid.org/0000-0002-4670-4658
dc.contributor.author	Jin, D https://orcid.org/0000-0003-1046-2666
dc.date.accessioned	2024-12-17T00:11:00Z
dc.date.available	2024-12-17T00:11:00Z
dc.date.issued	2024-03-19
dc.identifier.citation	Anal Chem, 2024, 96, (11), pp. 4430-4436
dc.identifier.issn	0003-2700
dc.identifier.issn	1520-6882
dc.identifier.uri	http://hdl.handle.net/10453/182604
dc.description.abstract	Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.
dc.format	Print-Electronic
dc.language	eng
dc.publisher	AMER CHEMICAL SOC
dc.relation	http://purl.org/au-research/grants/arc/FL210100180
dc.relation	http://purl.org/au-research/grants/arc/DE220100846
dc.relation.ispartof	Anal Chem
dc.relation.isbasedon	10.1021/acs.analchem.3c04530
dc.rights	info:eu-repo/semantics/closedAccess
dc.subject	0301 Analytical Chemistry, 0399 Other Chemical Sciences
dc.subject.classification	Analytical Chemistry
dc.subject.classification	3205 Medical biochemistry and metabolomics
dc.subject.classification	3401 Analytical chemistry
dc.subject.classification	4004 Chemical engineering
dc.subject.mesh	Lanthanoid Series Elements
dc.subject.mesh	In Situ Hybridization, Fluorescence
dc.subject.mesh	RNA
dc.subject.mesh	RNA, Messenger
dc.subject.mesh	Diagnostic Imaging
dc.subject.mesh	Lanthanoid Series Elements
dc.subject.mesh	RNA
dc.subject.mesh	RNA, Messenger
dc.subject.mesh	Diagnostic Imaging
dc.subject.mesh	In Situ Hybridization, Fluorescence
dc.subject.mesh	Lanthanoid Series Elements
dc.subject.mesh	In Situ Hybridization, Fluorescence
dc.subject.mesh	RNA
dc.subject.mesh	RNA, Messenger
dc.subject.mesh	Diagnostic Imaging
dc.title	Lanthanide Complex for Single-Molecule Fluorescent in Situ Hybridization and Background-Free Imaging.
dc.type	Journal Article
utslib.citation.volume	96
utslib.location.activity	United States
utslib.for	0301 Analytical Chemistry
utslib.for	0399 Other Chemical Sciences
pubs.organisational-group	University of Technology Sydney
pubs.organisational-group	University of Technology Sydney/Faculty of Science
pubs.organisational-group	University of Technology Sydney/Faculty of Science/School of Mathematical and Physical Sciences
pubs.organisational-group	University of Technology Sydney/UTS Groups
pubs.organisational-group	University of Technology Sydney/UTS Groups/Institute of Biomedical Materials and Devices (IBMD)
utslib.copyright.status	closed_access	*
dc.date.updated	2024-12-17T00:10:58Z
pubs.issue	11
pubs.publication-status	Published
pubs.volume	96
utslib.citation.issue	11

Abstract:

Traditional single-molecule fluorescence in situ hybridization (smFISH) methods for RNA detection often face sensitivity challenges due to the low fluorescence intensity of the probe. Also, short-lived autofluorescence complicates obtaining clear signals from tissue sections. In response, we have developed an smFISH probe using highly grafted lanthanide complexes to address both concentration quenching and autofluorescence background. Our approach involves an oligo PCR incorporating azide-dUTP, enabling conjugation with lanthanide complexes. This method has proven to be stable, convenient, and cost-effective. Notably, for the mRNA detection in SKBR3 cells, the lanthanide probe group exhibited 2.5 times higher luminescence intensity and detected 3 times more signal points in cells compared with the Cy3 group. Furthermore, we successfully applied the probe to image HER2 mRNA molecules in breast cancer FFPE tissue sections, achieving a 2.7-fold improvement in sensitivity compared to Cy3-based probes. These results emphasize the potential of time-resolved smFISH as a highly sensitive method for nucleic acid detection, free of background fluorescence interference.

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http://hdl.handle.net/10453/182604