Integrated Nanozymes with Nanoscale Proximity for in Vivo Neurochemical Monitoring in Living Brains Hanjun Cheng, †,∥ Lei Zhang, † Jian He, ‡ Wenjing Guo, † Zhengyang Zhou,* ,‡ Xuejin Zhang,* ,† Shuming Nie,* ,†,§ and Hui Wei* ,†,∥ † College of Engineering and Applied Sciences, Nanjing National Laboratory of Microstructures, Nanjing University, Nanjing, Jiangsu 210093, China ‡ Department of Radiology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, Jiangsu 210008, China § Department of Biomedical Engineering, Emory University, Atlanta, Georgia 30322, United States ∥ Collaborative Innovation Center of Chemistry for Life Sciences, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing, Jiangsu 210093, China * S Supporting Information ABSTRACT: Nanozymes, the nanostructures with enzymatic activities, have attracted considerable attention because, in comparison with natural enzymes, they offer the possibility of lowered cost, improved stability, and excellent recyclability. However, the specificity and catalytic activity of current nanozymes are still far lower than that of their natural counterparts, which in turn has limited their use such as in bioanalysis. To address these challenges, herein we report the design and development of integrated nanozymes (INAzymes) by simultaneously embedding two cascade catalysts (i.e., a molecular catalyst hemin and a natural enzyme glucose oxidase, GOx) inside zeolitic imidazolate framework (ZIF-8) nanostructures. Such integrated design endowed the INAzymes with major advantage in improved catalytic efficiency as the first enzymatic reaction occurred in close (nanoscale) proximity to the second enzyme, so products of the first reaction can be used immediately as substrates for the second reaction, thus overcoming the problems of diffusion-limited kinetics and product instability. The considerable high catalytic activity and stability enabled the INAzymes to efficiently draw a colorimetric detection of glucose with good sensitivity and selectivity. When facilitated with in vivo microdialysis, the INAzyme was successfully used for facile colorimetric visualization of cerebral glucose in the brain of living rats. Moreover, when further combined with microfluidic technology, an integrative INAzyme-based online in vivo analytical platform was constructed. The promising application of the platform was successfully illustrated by continuously monitoring the dynamic changes of striatum glucose in living rats’ brain following ischemia/reperfusion. This study developed a useful approach to not only functional nanomaterial design but also advanced platforms developments for diverse targets monitoring. N anozymes, the nanostructures with enzymatic activities, have attracted particular attention as emerging natural enzyme mimics. 1,2 Because of their unique characteristics relative to natural enzymes and even conventional artificial enzymes, nanozymes have been extensively explored for wide applications, such as in bioanalysis, 3−24 bioimaging, 25,26 and biomedicine. 27−31 For instance, researchers have recently achieved the selective glucose detection in serum by exploring the catalase-mimicking activity of cerium oxide nanoparticles. 5 Despite the substantial progress, the specificity and catalytic activity of currently developed nanozymes are still far lower than that of natural enzymes. 1,2,32−39 These shortcomings have in turn impeded the use of nanozymes, such as in bioanalysis (especially in in vivo analysis). Therefore, great efforts have been devoted to tackling these unmet challenges. The selectivity challenge could be partially overcome by combining a nanozyme with a natural enzyme, as demonstrated in our previous study. 4 In that case, iron oxide nanoparticle-based peroxidase mimic was combined with natural glucose oxidase (GOx) for colorimetric detection of glucose. However, the Received: March 11, 2016 Accepted: April 12, 2016 Article pubs.acs.org/ac © XXXX American Chemical Society A DOI: 10.1021/acs.analchem.6b00975 Anal. Chem. XXXX, XXX, XXX−XXX