**1. Introduction**

Molecular oxygen is a key component for maintaining physiological activities in almost all living systems. Hence oxygen sensing is of grea<sup>t</sup> importance to understand related physiological and pathological processes, such as cell respiration and tumor hypoxia [1,2]. In terms of in vivo or in vitro oxygen sensing, luminescence-based approaches are much more attractive with the merits of high sensitivity, high spatiotemporal resolution, and non-invasiveness [3,4]. Various luminescent oxygen probes have been developed so far. In comparison, polymeric nanoparticles incorporated with oxygen-sensitive dyes are the most competent probes, because the porous matrix can prevent the doped dyes from interference by ions or biomolecules in complicated physiological environments, while maintaining the free diffusion of oxygen molecules [5,6]. Ruthenium (Ru) (II)-based oxygen probes are believed to be more stable than other luminescent metal complexes [7].

For most of the established luminescent oxygen nanoprobes, however, quantum yield and oxygen sensitivity are more or less undermined by the concentration quenching of indicators or obstructed diffusion of oxygen by the matrix [8,9]. Very recently we have presented a type of luminescent nanoprobe based on Ru (II)-containing metallopolymers to bypass these issues [10]. Because the oxygen probes [Ru(bpy)3]<sup>2</sup>+ reside on the particle surface, the nanoprobes exhibit strong red luminescence free of aggregation-induced-quenching and high oxygen sensitivity. Another common disadvantage of nanoprobes is the most widely adopted single intensity-based detection modality, which could

be influenced by the incident lamp, detector, and uneven distribution of probes [11]. Although lifetime-based sensing approaches are immune from the influence of these drawbacks, the complexity and the demands on the optoelectronic components increase with the decreasing lifetimes. By contrast, luminescence ratiometric approaches (2-wavelength) allow for more accurate and robust detection with a built-in calibration. Lanthanide complexes have unique optical properties, such as narrow emission bands and large Stokes shift [12]. In particular, their luminescence is hardly influenced by oxygen due to the protection of 5s25p6 outer-shell. Given that lanthanide ions could be chelated to polymers [13–16], a ratiometric luminescent oxygen nanoprobe thus can be constructed by using lanthanide-containing and Ru-containing metallopolymers as the reference and probe dye, respectively.

In this work, a bipyridine-branched hydrophobic copolymer was utilized to chelate Tb (III) complex and Ru (II) complex, respectively, so that to produce oxygen-insensitive metallopolymer (Tb-Poly) and oxygen-sensitive metallopolymer (Ru-Poly). Herein the green emissive Tb-Poly is chosen under the consideration that the reference signal should be distinguished from the red sensing signal of Ru-Poly. Taking advantage of the two metallopolymers, biocompatible luminescent nanoparticles (NPs) were prepared by a nanoprecipitation method. The resulting NPs give a two-wavelength emission under 300 nm and 460 nm excitation in aqueous solution, the ratio of which is highly oxygen-dependent in the experimental conditions. Based on the ratiometric luminescence, intracellular oxygen in monolayer cells and three-dimensional multi-cellular tumor spheroids were both detected.

#### **2. Materials and Methods**
