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Microchimica Acta

, 186:424 | Cite as

Simultaneous determination of two phosphorylated p53 proteins in SCC-7 cells by an ICP-MS immunoassay using apoferritin-templated europium(III) and lutetium(III) phosphate nanoparticles as labels

  • Xiao Yin
  • Beibei Chen
  • Man He
  • Bin HuEmail author
Original Paper

Abstract

Phosphorylated p53 proteins are biomarkers with clinical utility for early diagnosis of cancer, but difficult to quantify. An inductively coupled plasma mass spectrometry (ICP-MS) based immunoassay is described here that uses uniform lanthanide nanoparticles (NPs) as elemental tags for the simultaneous determination of two phosphorylated p53 proteins. Apoferritin templated europium (Eu) phosphate (AFEP) NPs and apoferritin templated lutetium (Lu) phosphate (AFLP) NPs with 8 nm in diameter were used to label two phosphorylated p53 proteins at serine 15 and serine 392 sites (p-p5315 and p-p53392), respectively. The assay has a sandwich format, and p-p5315 and p-p53392 were first captured and then recognized by AFEP and AFLP NPs labelled antibodies, respectively. The Eu and Lu were then released from the immune complexes under acidic condition for ICP-MS measurement. The limits of detection for p-p5315 and p-p53392 are 200 and 20 pg·mL−1, with linear ranges of 0.5–20 and 0.05–20 ng·mL−1, respectively. The method was further applied to study the response of p-p5315 and p-p53392 in SCC-7 cells exposed to the natural carcinogen arsenite. A significant up-regulation of p-p5315 and p-p53392 can be observed when cells were exposed to arsenite at 5 μmol·L−1 level for 24 h.

Graphical abstract

Schematic presentation of the ICP-MS immunoassay using apoferritin templated europium (III) and lutetium (III) phosphate nanoparticles as labels for the simultaneous determination of two phosphorylated p53 proteins. Europium (Eu) phosphate nanoparticles (blue) and lutetium (Lu) phosphate nanoparticles (pink) were synthesized in the size-restricted cavity of apoferritin. They were further coupled with antibodies to prepare Eu and Lu labelled probes for p-p5315 (blue) and p-p53392 (pink), respectively. After formation of a a sandwich, the labelled Eu and Lu were dissociated in acid and then introduced to ICP-MS for the simultaneous determination of two phosphorylated p53 proteins p-p5315 (blue) and p-p53392 (pink).

Keywords

Cancer Biomarker Tumor suppressor Element label Carcinogen Arsenite Multi-analyte immunoassay Cell lysates 

Notes

Acknowledgements

This work is financially supported by the National Nature Science Foundation of China (Nos. 21775113, 21575107, 21575108, 21375097), the Science Fund for Creative Research Groups of NSFC (No. 20921062), the MOE of China, and the Large-scale Instrument and Equipment Sharing Foundation of Wuhan University (No. LF20191236).

Compliance with ethical standards

The author(s) declare that they have no competing interests.

Supplementary material

604_2019_3540_MOESM1_ESM.docx (536 kb)
ESM 1 (DOCX 535 kb)

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Copyright information

© Springer-Verlag GmbH Austria, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of ChemistryWuhan UniversityWuhanChina

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