# Sensor Micro and Nanoparticles for Microfluidic Application
## 2 Construction Principles of Sensor Particles
### Oxygen Sensing  
The concept of oxygen sensing and imaging is preferably based on the quenching of oxygen-sensitive dye luminescence by molecular oxygen. Due to quenching, the intensity of luminescence decreases, and changes in decay time occur. Intensity-based phosphorescence or fluorescence measurements are widely used for their simplicity, low cost and easily accessible equipment. However, these measurements are sensitive to excitation intensity and efficiency of detection system. Moreover, many other factors such as photobleaching, uneven dye distributions, scattering of excitation or emission light can strongly affect luminescence intensity. Therefore, it is necessary to apply referenced detection schemes--for example, the ratiometric method. During ratiometric fluorescence measurement, the intensities at two or more emission peaks of an oxygen-sensitive indicator and a reference dye are measured in order to detect changes in oxygen concentration. By applying this method, environment variations are reduced. Nonetheless, signal varieties due to photobleaching, leaching of fluorescent dye or Rayleigh scattering still have an impact on precise analyte measurement [53]. Another widely applied technique is fluorescence lifetime imaging, which is based on determination of the spatial distribution of fluorescence lifetimes at every pixel of the image. As the fluorescence lifetime of a fluorophore does not depend on its concentration, in the first approximation, the thickness of the sample, excitation intensity or photobleaching do not disturb the measurement seriously. The lifetime-based measurements provide information about the molecular environment of the fluorophore such as pH, concentration of oxygen and molecular binding. Fluorescence lifetime imaging techniques can be classified into frequency-domain and time-domain techniques. In the time-domain techniques, analyte is excited by short light and the fluorescence emission is recorded as a function of time. In the frequency domain, modulated source of light excites the sample and the fluorescence emission becomes modulated and phase-shifted from the excitation curve; therefore, fluorescence lifetimes are calculated from the observed phase-shift and modulation [54]. By combining the oxygen imaging with microfluidics systems, it is possible to visualize concentration gradients of analyte, evaluate concentrations, monitor chemical reactions and perform single cell analysis [53]. In order to investigate metabolism and growth of aerobic bacteria in droplet bioreactor systems, it is critical to monitor the oxygen concentration inside the droplets. Measurements of absolute oxygen concentration and optical density (OD) during incubation of _Escherichia coli_ and _M. smegmatis_ bacteria inside the droplets were performed and presented in the research of [PERSON] et al. [55]. In order to form an oxygen sensor, phosphorescent indicator dye (PtTPTPBF) was immobilized in core\(-\)shell structured poly-(styrene-block vinylpyrrolidone) (PSPVP) nanobeads. Due to the core\(-\)shell structure, sensor particles were dispersed in aqueous phase and oxygen-sensitive dyes were immobilizes in the lipophilic core of the nanobead. For phosphorescence frequency-domain lifetime measurements, the nanoparticles were excited by red light and due to large Stokes shift, particles emitted near-infrared (NIR) light. Fluorescence emission in the NIR region offered the ability to use PSPVP-PtTPBPF particles in biological samples because of minimized background fluorescence. The most significant effect was observed when a large quantity of oxygen was consumed during the exponential growth phase of bacterial colony, which resulted in the significantly decreased concentration of oxygen in the droplets. The absolute oxygen concentration was accurately determined with a resolution of 0.07-0.12 uM. Non-invasive, optical monitoring of oxygen concentration during bacterial cultivation in microfluidic droplet-based system was presented by [PERSON] et al. [6].