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

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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.