Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - AmpiDots (Exponential Amplification and Rapid Detection of miRNAs using DNA-Quantum Dot Bioconjugates for Disease Diagnostics)

Teaser

Accurate and timely diagnosis is an important step in the treatment of all diseases. Although traditional approaches to diagnostics have achieved much success in the past, the potential for advanced diagnostics is vastly bigger than what we have managed to unlock so far. The...

Summary

Accurate and timely diagnosis is an important step in the treatment of all diseases. Although traditional approaches to diagnostics have achieved much success in the past, the potential for advanced diagnostics is vastly bigger than what we have managed to unlock so far. The new and expanding frontier in diagnostics is molecular diagnostics, where specific molecules present in a patient\'s body can be correlated with a particular disease or health condition. In fact, the human body is filled with molecular signatures of disease, right from the beginning of infection or disease initiation. If we could read them, these molecular signatures would tell us much of what we need to know about the health status of an individual, and would inform us how we can intervene to correct disease states. However, these biomolecules are typically only present in very small quantities in the body, and thus they are hard to detect. Further, we might need to detect a large number of different biomolecules to paint a detailed picture of a patient\'s health status. To unlock the full potential of molecular diagnostics, it is imperative that we develop new technologies that allow sensitive detection of a range of biomolecules in patient samples.

Nanotechnology concerns the synthesis and application of materials in the size regime below 100 nanometers. A human hair is around 50 micrometers wide, which is 50’000 nanometers. Therefore, to reach the nanomaterial size threshold of 100 nanometers, we would need to split a hair 500 times width ways. Scientifically, there are various driving forces behind interest in nanomaterials, including the fact that many materials exhibit unique phenomena – such as intense fluorescence or magnetism – that are not present in bulk, when they are reduced in size into the nanoscale. Further, in the context of biological sciences, nanomaterials are in the same size regime as many biomolecules (e.g. proteins, nucleic acids), therefore they can be readily engineered to interact with biological systems. DNA nanotechnology concerns the use of DNA molecules as structural and functional units in nanomaterial constructs.

There is much potential for innovation in diagnostics by combining functional nanoparticles and enzymes. In scientific research, it has been observed that when the target (substrate) of enzymes are immobilized on nanoparticles, the reactions proceed differently than if the substrates are free in solution. The overall aim of the current project was to study the interaction of DNA nanomaterials with enzymes, working towards uncovering unique phenomena that could be harnessed in the detection of various molecular targets. The work was split into a fundamental side which looked at characterizing DNA nanomaterial–enzyme interactions, and an applied side that looked to create novel sensors and bioassays based on these interactions.

Work performed

The initial part of the project involved designing and synthesising a set of DNA-nanoparticle constructs to interact with specific nuclease enzymes. The DNA-nanoparticles consisted of a gold nanoparticle core that was decorated with many DNA strands (typically 25–50 units in length). These are termed spherical nucleic acids, or SNAs. The DNA strands were designed in such a way that they contained a recognition sequence for a nuclease enzyme, and that interaction with that enzyme would remove a fluorescent dye and lead to an increase in fluorescence as a signal of the interaction. In the enzyme experiments, the rate of digestion of the DNA was studied as a function of substrate and enzyme concentration, and compared directly to a ‘free’ system on DNA probes that were not bound to a particle. Thus, the study is looking to uncover the effects of immobilizing the nuclease substrates on a nanoparticle. This part of the work became the main focus of the project. We conducted a first-ever systematic study of exonuclease and endonuclease enzymes acting on SNAs. So far, this work has revealed that the certain enzyme can penetrate into, bind and cleave in the inner portion of the dense DNA corona around the SNA. This was previously considered to be improbable. Further, we have revealed the difference in reaction dynamics in a recycling system comparing a nanoparticle and free case.

The project involved developing microfluidic devices for the study of nanoparticle-enzyme interactions. A set of microfluidic chips were designed and manufactured for this purpose. An extension of this was a collaborative study looking at the use of reinforcement learning for the dynamic control of microfluidic devices. The rationale was that, as microfluidic devices typically need human intervention to maintain system stability over extended time periods (hours to days), it would be preferable to have a \'smart\' automated system that is able to replace human intervention. The developed reinforcement learning algorithms were able to attain superhuman performance in controlling and processing two test conditions (laminar flow and segmented flow), highlighting the utility of novel control algorithms for automated high-throughput microfluidic experimentation.

Final results

The work performed in this projects comprises a first-ever systematic study of how exonuclease and endonuclease enzymes interact with spherical nucleic acids, in target recycling and non-recycling formats. To date, observations in the literature have tended to result from studies focussed on assay development, and as such there are many unknowns left to be explored. Therefore, the current project results will contribute new knowledge to the field. We expect that technologies based around these materials will be important in the development future high performance medical diagnostic tools, with a positive societal impact in terms of both health- and lifespan.

Website & more info

More info: https://demellogroup.ethz.ch/en/people.