Autophagy is the body’s way of cleaning out damaged cells to allow for the regeneration of newer, healthier parts and components in cells. Autophagic flux is measured by blocking autophagosome activity and measuring the resulting build-up in light chain 3 (LC3) protein. Flux measurements can be correlated with neurodegeneration, most notably diseases such as Alzheimer’s disease or Parkinson’s disease, where specific proteins accumulate and lead to neuronal toxicity and ultimately cell death. Nathan Brown, a master’s student in the Department of Electric and Electronic Engineering is currently working with Prof Willie Perold, Department of Electrical & Electronic Engineering, Prof Ben Loos, CEO of Phagoflux, Department of Physiological Sciences, and Dr André du Toit to develop an autophagic flux method.
Project goals and plan
The research project intends to measure autophagic flux with an integrated microfluidic-electrochemical, multi-biomarker sensing device. To achieve the goals of this project, sequential subgoals must be achieved namely, repeatable and optimised measurement of LC3(-I), additional measurements of p62, GAPDH and Actin. Once these goals have been reached, the next steps in the development process will be assessed.
“From my research so far and from what I have, it doesn’t seem that measurement has been repeatable, and it definitely hasn’t been optimised. I’m going to need quite a stable, repeatable and optimised measurement of LC3 moving forward. So, to achieve this repeatable measurement I’m currently working towards making an automated microfluidic experimental system,” says Nathan.
Measurement of LC3
Even though some measurement processes for LC3 have already been established, these measurements have not had high repeatability or been optimised. “To achieve repeatable and optimised measurements of LC3, I am creating an automated microfluidic system to conduct LC3 measurement experiments,” explains Nathan.
Using this automated microfluidic experiment system should significantly increase repeatability by removing human involvement and so allow the creation of baselines and enabling precise control of experimental variables such as volume, incubation time and so forth. It will further allow for the discovery of optimised conditions by enabling parameter sweeps to be conducted that were too large to be done manually. Nathan explains that this system will allow him to “development knowledge in the microfluidics design and implementation by using, for example, COMSOL and a laser cutter as a simple first step before ‘attempting’ to make more complicated integrated microfluidic-electrochemical systems.
Experimental system: Current challenges
Although progress has been substantial there are certain challenges: While functionalising with the microfluidic system should improve repeatability, 3MPA requires precise placement only on the working electrode (WE). However, droplets are not stable on the hydrophobic WE as both the PMMA chamber and exposed ceramic layer around the WE are hydrophilic.
There are a few options to solve these issues which will be evaluated in due course. These options include applying hydrophobic coating on the hydrophilic surfaces, COMSOL designing an improved fluidic structure, or avoiding the problem entirely by functionalising electrodes outside of microfluidics.
The problem of droplet control has subsequently been addressed by exploiting the phenomenon of meniscus pinning, and substantial progress has now been made towards the manufacture and integration of the various subsystems. All the fluidic channels and valves involved are included on a single PMMA microfluidic board. The syringe pumps and solution reservoirs are connected to this board with FEP tubing and custom laser cut connectors. The SPE is clamped to the other end of the fluidic board, and base functionality for controlling the incubation chamber has been validated. Initial voltammetric measurements have been taken with a potentiostat, which completed the integration of the final subsystem and allows us to finally begin with protein measurements.
Summary and going forward
The overall idea of this project is to create a multichannel integrated microfluidic-electrochemical measurement device for autophagic flux. The immediate goals are (1) reaching the repeatable and optimised measurement of LC3 with the (2) additional measurements of p62, (3) GAPDH and Actin. The first step is to develop an automated experiment system to achieve goal 1 which will ultimately assist in achieving later goals. Nathan is currently busy implementing the microfluids of this experiment system whereafter he will integrate the rest of the system and start collecting data.
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