Objective Diabetes mellitus is a chronic and noncommunicable disease with complications in the retina, heart, kidney, and neural system. The effective monitoring of blood glucose level is crucial in the prevention, diagnosis, and management of diabetes. Compared with single-point detection, a continuous glucose monitoring system can track the blood glucose fluctuation and help in predicting the trend of blood glucose change. Recently, various continuous glucose monitoring systems have been developed. The most widely used monitoring modality is electrochemical sensors, which collect glucose information in the interstitial fluid using an implanted enzyme-immobilized electrode. However, electrochemical sensors have some issues, including the limited glucose monitoring time and the risk of infection. Conversely, transdermal detection-based optical sensors exhibit prolonged service time and decreased risk of infection. Luminescent nanoparticles have shown great potential in biological applications because of their high brightness, high photostability, and good biocompatibility. Here, we developed a continuous glucose monitoring system based on a visible-light-excited nanoparticle transducer. We experimentally demonstrated that the nanoparticle transducer is promising for sensitive glucose detection. The visible-light-excited transducer shows potential in long-term and high-frequency monitoring in practical applications with reduced side effects compared with ultraviolet radiation. Methods The nanoparticles were prepared using a visible-light-excited fluorescent molecule and the oxygen-sensitive phosphorescent dye via the reprecipitation method. The resulting nanoparticles were characterized via UV-Vis absorption spectra, transition electron microscopy ( TEM) , and dynamic light scattering ( DLS) measurements. The glucose-sensitive nanoparticle transducer was formed by modifying glucose oxidase onto the surface of nanoparticles via EDC-catalyzed bioconjugation. The successful bioconjugation of oxygen-consuming enzyme onto the nanoparticle was characterized by the change in hydrodynamic diameters and zeta-potentials. The biocompatibility of the nanoparticles was characterized through cytotoxicity experiment. The glucose sensitivity of the nanoparticle transducer was examined by measuring the emission spectra under different glucose concentrations (0-20 mmol/L) . The intracellular glucose sensing of the nanoparticle transducer was performed on MCF-7 cells. The MCF-7 cells were incubated with the nanoparticle transducer first in a sugar-free medium. Additional glucose solution was introduced with the final concentrations at 20 mmol/L. The luminescence images under different glucose concentrations (0 and 20 mmol/L) were captured. We investigated the in vivo glucose monitoring performance of the nanoparticle transducer. The nanoparticle-GOx transducer (50 mu g/mL) was subcutaneously implanted in the lower back of mice under anesthesia. The blood glucose concentrations of the mice were elevated by the intraperitoneal injection of glucose solutions (1 mol/L, in Milli-Q water) . Subsequently, blood samples were collected from the tail of mice, and the blood glucose concentrations were tested using a commercial glucometer. Using a small animal biophotonic imaging system, the luminescence images of the subcutaneously implanted nanoparticle-GOx transducer were collected. The luminescence intensity of the implanted nanoparticle transducer was measured and compared with blood glucose concentrations. The mice with the intraperitoneal injection of PBS were adopted as the control group. Results and Discussions The oxygen-sensitive nanoparticle comprises a fluorescent molecule DPBF, an oxygen-sensitive phosphorescent dye PdTFPP, and a functional polymer PSMA. The resulting nanoparticles have a hydrodynamic diameter of 18 nm, as indicated by the TEM and DLS results. The successful bioconjugation of glucose oxidase onto the nanoparticle surface was characterized by the increased hydrodynamic diameters and zeta-potentials. According to the spectroscopic experiments, the phosphorescence intensity ( similar to 672 nm) of the nanoparticle-GOx transducer increased as the glucose concentration increased, whereas the fluorescence intensity ( similar to 490 nm) remained unchanged. The designed ratiometric sensing system can help in eliminating the luminescence fluctuations caused by the variation in excitation intensity and environmental conditions. The nanoparticle-GOx transducers exhibited a fast response time to distinguish different glucose concentrations. The luminescence spectra of the nanoparticle-GOx transducers under different glucose concentrations were measured within 10 min after adding glucose to the solution. A good correlation was exhibited between the luminescence intensity of the nanoparticle-GOx transducers and glucose concentrations. The cell viability of the MCF-7 cells did not change considerably after incubating with the nanoparticles at different concentrations. The results indicated that the nanoparticles are biocompatible for the following intracellular glucose sensing experiments and in vivo glucose monitoring experiments. The nanoparticle-GOx transducer exhibited a reversible response to glucose, and the monitoring performance remained unchanged for more than 10 repetitive tests. The nanoparticle-GOx transducer exhibited excellent photostability against hydrogen peroxide and free radicals. The reversible response and excellent photostability enabled the stable and long-term continuous glucose monitoring. After adding glucose, the luminescence intensity of the internalized nanoparticle transducers by the cells was obviously enhanced, indicating that the nanoparticle transducer has the potential for intracellular glucose sensing. For in vivo glucose monitoring, we collected the luminescence images of subcutaneously implanted nanoparticle transducers. The transdermal detection of the nanoparticle transducers was achieved because of its high brightness. After the intraperitoneal injection of glucose solution, the luminescence intensity of the subcutaneously implanted transducers increased with the increased blood glucose concentrations, whereas in the control group, the luminescence intensity and blood glucose concentrations remain unchanged after the intraperitoneal injection of PBS. These results indicate that the luminescent nanoparticle transducers are promising for in vivo continuous glucose sensing. Conclusions We developed a continuous glucose monitoring system based on a visible-light-excited luminescent nanoparticle transducer. The nanoparticle transducer can be used for the transdermal detection of blood glucose because of its high luminescence brightness. We demonstrated in vitro cellular glucose sensing and in vivo glucose monitoring in animal models. With the ratiometric sensing system, the signal fluctuations caused by the variation in excitation intensity and environmental conditions would be eliminated. The visible-light-excited transducer can also avoid the side effects induced by ultraviolet radiation, indicating the potential for long-term and high-frequency monitoring in practical applications.
