The problem of fast and qualitative bioassay is relevant today. There are traditional methods of drinking water, industrial waste water and biological fluids bioassay, based on the experimental animals and the protozoa organisms. However, their usage requires laboratory conditions. These techniques are difficult to adapt for personal biodiagnostics devices. Such bioassay also has a problem of high error associated with individuality of the test organisms.
One way to overcome these problems is to use enzyme-based bioassay methods based on bioluminescence reaction catalyzed by luciferase and NAD(P)H:FMN-oxidoreductase that were extracted from luminescent bacteria Vibreo harvery. By measuring the catalytic activity of pollutant sensitive enzymes, it has become possible to draw conclusions about the degree of contamination of natural, industrial waters and biological fluids. Compared to traditional analysis methods, in many cases the use of bioluminescent techniques along with increased accuracy and sensitivity of analysis can reduce the cost by approximately 70% and significantly reduce the duration of analysis. But today there is a lack of a suitable set of techniques for automation and implementation of enzymatic bioluminescent method of rapid bioassay of natural, industrial waters and biological fluids
This method needs to be automated and implemented in the form of disposable chips with immobilized components of the test system and a in a form of continuous flow diagnostic systems based on high frequency droplet generators in which the enzymes of the bioluminescent test system are aggregated to nanodiamonds and reactions will take place in isolated nanoliter volumes of samples.
This project is aimed for creation of an automated high-sensitivity analytical platform for enzymatic bioluminescent test of natural, industrial water and biological fluids using microfluidic technology and functionalized nanoparticles. Luciferase microfluidic platform consists of a set of microfluidic components (drive, valve, mixer, etc.), biological component and signal detection system. This set of components is easily combined with each other in the process of production of miniaturized biosensors.
Nanodiamonds will be the essential part of signal detection system in droplets, because they allow to manipulate active compounds in flows of microfluidic chips.
Scientific novelty of the research lies in the theoretical modeling and conducting of experiments in which signal is generated by luciferase and amplified by nanoparticles in droplets. That allows to create a new method for rapid testing of physiological fluids and water. The engineering novelty lies in designing, manufacturing and debugging of chips based on methods of low-temperature sealing of chips and
immobilization of proteins that allows to preserve enzyme activity and solubilization of nanoparticles that allows to control these enzymes in chips. Technological novelty lies in the use of the collective effects of the interaction of diamond nanoparticles with molecules of proteins in biomodule of droplet-reactors.
When implementing these approaches a microfluidic device for the analytical assessment of the contamination degree of the samples and the study of enzymatic reactions in the droplet will be created.
Since with bienzyme luciferase system such task of measurements automation have not been conducted before, the development of such a platform is a completely new challenge.