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Mikrochim Acta. 2019 May 10;186(6):344. doi: 10.1007/s00604-019-3476-8.

Immunodetection and counting of circulating tumor cells (HepG2) by combining gold nanoparticle labeling, rolling circle amplification and ICP-MS detection of gold.

Author information

1
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China.
2
Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan, 430072, China. binhu@whu.edu.cn.

Abstract

A method is described for counting circulating tumor cells (CTCs). It is making use of inductively coupled plasma mass spectrometry (ICP-MS) along with a dual amplification strategy by combining rolling circle amplification (RCA) and gold nanoparticle (Au NP) labeling. HepG2 cells, as a representative CTC line, were captured by anti-epithelial cellular adhesion molecule (EpCAM) immobilized on a microplate, then specifically labeled with biotinylated anti-asialoglycoprotein receptor (ASGPR). Taking streptavidin (SA) as the bridge, the biotinylated RCA primer was conjugated to HepG2 cells. When the RCA reaction was triggered, long ssDNA with tandem repeats generated on the cell surface. Then, Au NP functionalized detection DNA (signal probes) was added to hybridize with the ssDNA. After removing the redundant signal probes, Au NPs conjugated on target HepG2 cells were subjected to ICP-MS detection. By adopting such a dual amplification strategy, a 756-fold improvement in sensitivity is accomplished compared to the method involving only Au NP labeling without RCA. The limit of detection is as low as 3 HepG2 cells (15 cell mL-1) which is the lowest LOD in ICP-MS based methods for cell counting. Besides, the method provides good selectivity, a wide linear range of 10-1000 HepG2 cells (50-5000 cells mL-1), and relative standard deviations of 6.3% (nā€‰=ā€‰7; 50 HepG2 cells (250 cells mL-1)). The method was successfully applied to HepG2 cell counting in spiked human blood samples and gave good recoveries. Graphical abstract Schematic presentation of an ICP-MS based immunoassay for the sensitive circulating tumor cells counting by combining rolling circle amplification (RCA) with gold nanoparticle (Au NP) labeling. ICP-MS: inductively coupled plasma mass spectrometry; ASGPR: asialoglycoprotein receptor; EpCAM: epithelial cellular adhesion molecule.

KEYWORDS:

Elemental labeling strategy; Elemental tag; Human blood sample; Inductively coupled plasma mass spectrometry; Sensitivity improvement

PMID:
31076917
DOI:
10.1007/s00604-019-3476-8

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