Infoscience

Thesis

Multiscattering-enhanced optical probes for biosensing

Although reactive oxygen species (ROS) are an inescapable part of every living organism, we are only beginning to understand their functions and mechanics. As of now, we know that ROS are involved in cell signalling, stress-response and defence against pathogens. New theories suggest that ROS also play an important role in various diseases and aging. However, further research is hindered by the fact that ROS are small and highly reactive molecules, making them very difficult to detect. Therefore, we need new sensitive and biocompatible tools with the ability to perform continuous measurements of ROS. To realise such a sensor, I first developed an original scheme for sensitive absorption measurements – multiscattering-enhanced optical absorption or MEAS. MEAS benefits from the advantages of conventional absorption spectroscopy and relies on extending the optical path of light through the sensing volume by creating a multiscattering configuration. This way, higher sensitivity and lower limit of detection, compared to those of conventional absorption spectroscopy, were achieved. I further studied numerically the energy transfer from light into the absorbing background in the presence of a ran-dom medium. I discovered that there is a regime of maximal absorption in such a system, which corresponds to a specific filling factor of random medium. Having understood principles involved in sensing using random media, I designed and fabricated biocompatible and miniature optical probes: picomoles of the protein cytochrome c printed inside a porous matrix. The refractive index of porous membrane exhibits spatial variations leading to multiscattering of light and consequently to enhanced optical path lengths. Such probes can trace the dynamics of hydrogen peroxide (H2O2 – the most stable ROS) with a detection limit of 40 nM. Next, I have incorporated the developed optical probes into a portable oxidative stress sensor – a stand-alone device specifically designed for efficient and easy analysis in the field.I further extended multiscattering-enhanced optical probes to continuous measurements of glucose and lactate – important metabolites in cells. The detection scheme is based on cytochrome c in conjunction with the respective enzymes. I obtained limits of detection of 240 nM and 110 nM for lactate and glucose, respectively. These values are at least one order of magnitude lower than the state of the art. Overall, the developed multifunctional detection scheme provides a powerful tool to study cellular biochemical processes. After realising very sensitive optical probes with the possibility of continuous measurements, I used the developed portable sensor to study the dynamics of H2O2 released by C.reinhardtii under stress. Besides the tremendous importance for fundamental ROS biology, this is also a way to assess and compare the toxicity of different engineered nanomaterials (ENMs). In this context, I fabricated a multi-layered microfluidic chip with integrated microfluidic valves and microsieves that allows carrying out experiments with complex sequences of analytes exposure, mixing and rinsing. The response of algae to two Cd-based toxicants (ionic Cd and CdSe/ZnS quantum dots) was studied. As a result, I showed quantitative dynamics of extracellular H2O2, the recovery of cell homeostasis after short-term stress and the cumulative nature of two consecutive exposures.

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