Organosilica Nanosensors for Monitoring Spatiotemporal Changes in Oxygen Levels in Bacterial Cultures.

Oxygen plays a central role in aerobic metabolism, and while many approaches have been developed to measure oxygen concentration in biological environments over time, monitoring spatiotemporal changes in dissolved oxygen levels remains challenging. To address this, we developed a ratiometric core-shell organosilica nanosensor for continuous, real-time optical monitoring of oxygen levels in biological environments. The nanosensors demonstrate good steady state characteristics (KpSV = 0.40 L/mg, R2 = 0.95) and respond reversibly to changes in oxygen concentration in buffered solutions and report similar oxygen level changes in response to bacterial cell growth (Escherichia coli) in comparison to a commercial bulk optode-based sensing film. We further demonstrated that the oxygen nanosensors could be distributed within a growing culture of E. coli and used to record oxygen levels over time and in different locations within a static culture, opening the possibility of spatiotemporal monitoring in complex biological systems.

Review: Nanomaterials for Reactive Oxygen Species Detection and Monitoring in Biological Environments.

Reactive oxygen species (ROS) and dissolved oxygen play key roles across many biological processes, and fluorescent stains and dyes are the primary tools used to quantify these species in vitro. However, spatio-temporal monitoring of ROS and dissolved oxygen in biological systems are challenging due to issues including poor photostability, lack of reversibility, and rapid off-site diffusion. In particular, ROS monitoring is hindered by the short lifetime of ROS molecules and their low abundance. The combination of nanomaterials and fluorescent detection has led to new opportunities for development of imaging probes, sensors, and theranostic products, because the scaffolds lead to improved optical properties, tuneable interactions with cells and media, and ratiometric sensing robust to environmental drift. In this review, we aim to critically assess and highlight recent development in nanosensors and nanomaterials used for the detection of oxygen and ROS in biological systems, and their future potential use as diagnosis tools.

Stability and Performance Study of Fluorescent Organosilica pH Nanosensors.

Long-term stability and function are key challenges for optical nanosensors operating in complex biological environments. While much focus is rightly placed on issues related to specificity, sensitivity, reversibility, and response time, many nanosensors are not capable of transducing accurate results over prolonged time periods. Sensors could fail over time due to the degradation of scaffold material, degradation of signaling dyes and components, or a combination of both. It is critical to investigate how such degradative processes affect sensor output, as the consequences could be severe. Herein, we used fluorescent core-shell organosilica pH nanosensors as a model system, incubating them in a range of common aqueous solutions over time at different temperatures, and then searched for changes in fluorescence signal, particle size, and evidence of silica degradation. We found that these ratiometric nanosensors produced stable optical signals after aging for 30 days at 37 °C in standard saline buffers with and without 10% fetal bovine serum, and without any evidence of material degradation. Next, we evaluated their performance as real-time pH nanosensors in bacterial suspension cultures, observing a close agreement with a pH electrode for control nanosensors, yet observing obvious deviations in signal based on the aging conditions. The results show that while the organosilica scaffold does not degrade appreciably over time, careful selection of dyes and further systematic investigations into the effects of salt and protein levels are required to realize long-term stable nanosensors.

Modified Organosilica Core-Shell Nanoparticles for Stable pH Sensing in Biological Solutions.

Continuous monitoring using nanoparticle-based sensors has been successfully employed in complex biological systems, yet the sensors still suffer from poor long-term stability partially because of the scaffold materials chosen to date. Organosilica core-shell nanoparticles containing a mixture of covalently incorporated pH-sensitive (shell) and pH-insensitive (core) fluorophores is presented as a continuous pH sensor for application in biological media. In contrast to previous studies focusing on similar materials, we sought to investigate the sensor characteristics (dynamic range, sensitivity, response time, stability) as a function of material properties. The ratio of the fluorescence intensities at specific wavelengths was found to be highly sensitive to pH over a physiologically relevant range (4.5-8) with a response time of <100 ms, significantly faster than that of previously reported response times using silica-based particles. Particles produced stable, pH-specific signals when stored at room temperature for more than 80 days. Finally, we demonstrated that the nanosensors successfully monitored the pH of a bacterial culture over 15 h and that pH changes in the skin of mouse cadavers could also be observed via in vivo fluorescence imaging following subcutaneous injection. The understanding gained from linking sensor characteristics and material properties will inform the next generation of optical nanosensors for continuous-monitoring applications.