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

Abstract

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.

Keywords: NIR spectroscopy; Nanomedicine; Photodynamic Therapy; Radiotherapy; Singlet oxygen detection.