Intracellular dark-field imaging of ATP and photothermal therapy using a colorimetric assay based on gold nanoparticle aggregation via tetrazine/trans-cyclooctene cycloaddition

  • Fei Liu
  • Yingshu GuoEmail author
  • Yinhua Hu
  • Xiaoru Zhang
  • Xiangjiang Zheng
Research Paper


In this study, we developed a colorimetric ATP assay based on the ATP-induced aggregation of Au nanoparticles (AuNPs). This aggregation modified the local surface plasmon resonance (LSPR) of the AuNPs, which was used to detect and localize ATP in cells via dark-field imaging. The AuNP aggregation process involved the reaction of two types of functionalized AuNPs with each other: tetrazine-modified AuNPs (Au3-N4) and asymmetrically functionalized trans-cyclooctene-modified AuNPs (Au1-(E)-cyclooctene). This cycloaddition reaction occurs without the need for a catalyst such as the Cu ions that are used in the “click” reactions often employed in assays of this type. Initially, we asymmetrically functionalized both types of AuNPs and let them dimerize, which permitted us to explore the resulting wavelength shift in the LSPR of the AuNPs. Then, to facilitate the specific recognition of ATP, a designed DNA (DNA1) containing an ATP aptamer sequence was attached to carboxyl polystyrene microbeads (MBs). After attaching a different DNA (DNA2, which hybridizes with DNA1) to Au1-(E)-cyclooctene, the assay probe MB/DNA1/DNA2/Au1-(E)-cyclooctene (MB/Au1) was generated. While bound to MB/DNA1, the DNA2/Au1-(E)-cyclooctene cannot react with Au3-N4 due to steric hindrance from the MB. However, in the presence of ATP, the probe MB/Au1 dissociates, and the resulting free DNA2/Au1-(E)-cyclooctene can then react with the Au3-N4, leading to the formation of AuNP aggregates. Dark-field microscopy (DFM) images showed that the LSPR of the AuNPs shifted from the green region (AuNP monomers) to the orange-red region (AuNP aggregates) in the presence of intracellular ATP. Moreover, the AuNP aggregates were found to exhibit significant photothermal effects under 808-nm laser irradiation. Upon introducing the probe MB/Au1 and Au3-N4 into HeLa cells in vitro and in vivo, and then irradiating the cells with a 808-nm NIR laser, the resulting AuNP aggregates showed promising photothermal cancer therapy performance. This assay therefore has the potential to be widely used for the identification and determination of nanoparticles in biological DFM and in tumor theranostics.

Graphical abstract


Dark-field imaging Intracellular ATP Photothermal therapy AuNP aggregates Tetrazine/trans-cyclooctene cycloaddition 


Funding information

This work was supported by the National Natural Science Foundation of China (21575056), the Natural Science Foundation of Shandong Province of China, (ZR2016JL010), and the Primary Research and Development Plan of Shandong Province (2018GSF118172).

Compliance with ethical standards

Conflict of interests

The authors declare that there is no conflict of interest.

Ethics approval and consent to participate

All animal experimental procedures and techniques were approved by the Animal Ethics Committee of East China Normal University, and methods were carried out in accordance with the approved guidelines and laws.

Supplementary material

216_2019_1966_MOESM1_ESM.pdf (1 mb)
ESM 1 (PDF 1029 kb)


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Copyright information

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

Authors and Affiliations

  • Fei Liu
    • 1
    • 2
  • Yingshu Guo
    • 2
    Email author
  • Yinhua Hu
    • 2
  • Xiaoru Zhang
    • 3
  • Xiangjiang Zheng
    • 2
  1. 1.College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Institute of Biomedical SciencesShandong Normal UniversityJinanChina
  2. 2.Collaborative Innovation Center of Tumor Marker Detection Technology, Equipment and Diagnosis-Therapy Integration in Universities of Shandong, Shandong Province Key Laboratory of Detection Technology for Tumor Markers, Shusheng Zhang Innovation Studio for Science and Technology Leader of Shandong Province, School of Chemistry and Chemical EngineeringLinyi UniversityLinyiChina
  3. 3.Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, College of Chemistry and Molecular EngineeringQingdao University of Science and TechnologyQingdaoChina

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