Spatial and temporal distributions of metal ions in vitro and in vivo are crucial in our understanding of the roles of metal ions in biological systems, and yet there is a very limited number of methods to probe metal ions with high space and time resolution, especially in vivo. To overcome this limitation, we report a Zn2+-specific near infrared (NIR) DNAzyme nanoprobe for real-time metal ion tracking with spatiotemporal control in early embryos and larvae of zebrafish. By conjugating photocaged DNAzymes onto lanthanide-doped upconversion nanoparticles (UCNPs), we have achieved upconversion of a deep tissue penetrating NIR 980 nm light into 365 nm emission. The UV photon then efficiently photo-decages a substrate strand containing a nitrobenzyl group at the 2’-OH of adenosine ribonucleotide, allowing enzymatic cleavage by a complementary DNA strand containing a Zn2+-selective DNAzyme. The product containing a visible FAM fluorophore that is initially quenched by BHQ1 and Dabcyl quenchers, is released after cleavage, resulting in higher fluorescent signals. The DNAzyme-UCNP probe enables Zn2+ sensing by exciting in the NIR biological imaging window in both living cells and zebrafish embryos, and detecting in the visible region. This report introduces a platform that can be used to understand the Zn2+ distribution with spatiotemporal control, thereby giving insights into the dynamical Zn2+ ion distribution in intracellular and in vivo models.

The quality and application of super-resolution fluorescence imaging greatly lie in the dyes’ properties, including photostability, brightness, and Stokes shift. Here we report a synergistic strategy to simultaneously improve such properties of regular fluorophores. Introduction of quinoxaline motif with fine-tuned electron density to conventional rhodamines generates new dyes with vibration structure and inhibited twisted-intramolecular-charge-transfer (TICT) formation synchronously, thus increasing the brightness and photostability while enlarging Stokes shift. The new fluorophore YL578 exhibits around twofold greater brightness and Stokes shift than its parental fluorophore, Rhodamine B. Importantly, in Stimulated Emission Depletion (STED) microscopy, YL578 derived probe possesses a superior photostability and thus renders threefold more frames than carbopyronine based probes (CPY-Halo and 580CP-Halo), known as photostable fluorophores for STED imaging. Furthermore, the strategy is well generalized to offer a new class of bright and photostable fluorescent probes with long Stokes shift (up to 136 nm) for bioimaging and biosensing.

RNA-cleaving DNAzymes have been demonstrated as a promising platform for sensing metal ions. However, the poor biological imaging performance of RNA-cleaving DNAzyme-based fluorescent probes has limited their intracellular applications. Compared with traditional one-photon fluorescence imaging, two-photon (TP) fluorescent probes have shown advantages such as increased penetration depth, lower tissue autofluorescence, and reduced photodamage. Herein, for the first time, we developed an RNA-cleaving DNAzyme-based TP imaging probe (TP-8-17ES-AuNP) for Zn detection in living cells by modifying a Zn-specific DNAzyme (8-17) with a TP fluorophore (TP-8-17ES) and using gold nanoparticles (AuNPs) for intracellular delivery. The modified TP-8-17ES exhibits good two-photon properties and excellent photostability. For the TP-8-17ES-AuNP, in the absence of Zn, the TP fluorophore is quenched by both AuNPs and the molecular quencher. Only in the presence of Zn does the DNAzyme cleave the TP fluorophore-labeled substrate strand, resulting in fluorescence enhancement and TP imaging. Such probe shows remarkable selectivity of Zn over other metal ions existing in the biological environment. Benefiting from the labeled TP fluorophore, the near-infrared (NIR) excited probe has the capability of TP imaging of Zn in living cells and tissue with a deep tissue penetration up to 160 μm. This method can be generally applied to detect other metal ions in biological systems under TP imaging with higher tissue penetration ability and lower phototoxicity.

Cell membrane–targeted bioimaging is a prerequisite for studying the roles of membrane-associated biomolecules in various physiological and pathological processes. However, long-term in situ bioimaging on the cell membrane with conventional fluorescent probes leads to diffusion into cells from the membrane surface. Therefore, we herein proposed a de novo strategy to construct an antidiffusion probe by integrating a fluorochrome characterized by strong hydrophobicity and low lipophilicity, with an enzyme substrate to meet this challenge. This precipitating fluorochrome HYPQ was designed by conjugating the traditionally strong hydrophobic solid-state fluorochrome 6-chloro–2-(2-hydroxyphenyl) quinazolin-4(3H)-one (HPQ) with a 2-(2-methyl–4H-chromen–4-ylidene) malononitrile group to obtain closer stacking to lower lipophilicity and elongate emission to the far-red to near-infrared wavelength. As proof-of-concept, the membrane-associated enzyme γ-glutamyltranspeptidase (GGT) was selected as a model enzyme to design the antidiffusion probe HYPQG. Then, benefiting from the precipitating and stable signal properties of HYPQ, in situ imaging of GGT on the membrane was successfully realized. Moreover, after HYPQG was activated by GGT, the fluorescence signal on the cell membrane remained unchanged, with incubation time even extending to 6 h, which is significant for in situ monitoring of enzymatic activity. In vivo testing subsequently showed that the tumor region could be accurately defined by this probe after long-term in situ imaging of tumor-bearing mice. The excellent performance of HYPQ indicates that it may be an ideal alternative for constructing universal antidiffusion fluorescent probes, potentially providing an efficient tool for accurate imaging-guided surgery in the future.