A portable NIR fluorimeter directly quantifies singlet oxygen generated by nanostructures for Photodynamic Therapy

This paper reports on the setting up and calibration of a portable NIR fluorimeter specifically developed for quantitative direct detection of the highly reactive singlet oxygen (1O2) chemical specie, of great importance in Photodynamic therapies. This quantification relies on the measurement of fluorescence emission of 1O2, which is peaked in the near-infrared (NIR) at λ=1270nm. In recent years, several nanostructures capable of generating reactive oxygen species (ROS) when activated by penetrating radiation (X-rays, NIR light) have been developed to apply Photodynamic Therapy (PDT) to tumours in deep tissue, where visible light cannot penetrate. A bottleneck in the characterization of these nanostructures is the lack of a fast and reliable technique to quantitatively assess their performances in generating ROS, and in particular 1O2. For instance, the widely used PDT “Singlet Oxygen Sensor Green” kit suffers from self-activation under X-ray irradiation. To solve this difficulty, we propose here direct detection of 1O2 by spectroscopic means, using an apparatus developed by us around a recent thermoelectrically-cooled InGaAs single photon avalanche photodiode (SPAD). The SPAD is coupled to a custom-made integrating sphere designed for use under irradiation with high-energy X-ray beams from clinical Radiotherapy sources. We determine the detection threshold for our apparatus, which turns to be ∼9·1081O2 in realistic experimental condition and for measurements extending to 1 min of integration. After calibrations on standard photosensitizers, we demonstrate the potentiality of this instrument characterizing some photosensitizing nanostructures developed by us.