In summary, our approach of integrating hydrophobic persistent luminescence nanoparticles into an optode-based nanosensor allowed for autofluorescence-free measurements in biological samples. An advantage of the ion-selective optode approach is the ability to tune dynamic range and selectivity depending on the sensing components in the polymeric matrix . Utilizing this modularity enabled us to fabricate persistent luminescence nanosensors for five different biologically relevant analytes. While long-lifetime emissions have been used for the quantification of O2 using phosphorescent lifetime imaging or other custom microscopes/CCD camera setups , the systems are often expensive or difficult to build for the end user. We showed that the ratiometric O2 PLNs can be used in a standard plate reader for facile time-resolved monitoring of O2 consumption in a high throughput manner. Overall, we anticipate that the PLNP nanocomposite can be used as a multifunctional A.