Team:RHS-Calgary/Engineering
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Engineering Success
iterative design in our project
In our experimentation thus far, there have been minimal occasions where we have encountered non-trivial obstances. However, one main instance in which we faced such scenarios was when we conducted our third main experiment. In this experiment, the system was seen to behave unexpectedly a number of times as it deviated from our initially proposed hypothesis. This experiment was conducted in two 24-well plates, with each plate containing two identical trials of each positive and negative control(This is shown in Figure 2, below). Our initial hypothesis was as follows: Wells containing trials 1 through 7 are negative controls and will thus not yield any change in appearance; However, wells containing trials 8 and 9 are positive controls and will in fact express a visible colour change from yellow to purple. While our team was confident that the above hypothesis would accurately represent our desired results, we faced some rather concerning results instead. We found that trials one through three, in both plates and in each duplicate trial, failed every time in that they expressed an obvious colour change from yellow to purple, though the expected result was for there to be no color change.
Immediately after observing these results, our wet lab team diligently began to work on creating yet another hypothesis as to why our negative controls may have failed. Through upholding a fundamental understanding of the reaction in play, we discussed that a colour change can only occur when components of the Working Reagent solution are exposed to Hydrogen Peroxide. Using various bioinformatic, biochemical, and biological research articles, we narrowed down the possibilities regarding where the possible source of contamination arose from, hence the creation of another hypothesis: When alpha-D-Glucose undergoes oxidation by GOx, a reaction takes place that can produce a byproduct of vaporous Hydrogen Peroxide. Due to H2O2’s highly volatile nature, it can react with the components of the colorimetric assay kit in surrounding wells containing negative controls, henceforth producing false-positive results.
It is important to note that the negative controls affected were immediately next to the wells containing the positive controls in the plate. This observation is what led us to the conclusion that trace amounts of gaseous Hydrogen Peroxide were being produced as a byproduct of the reaction, thus only contaminating the wells directly surrounding the positive controls. In order to approach the system which yielded non trivial results, we determined that the wells needed to be isolated from the surrounding environment and from each other. The Wet Lab team approached this by sealing each plate with a thin sheet of parafilm wax, allowing it to be airtight enough to eliminate contamination through H2O2 vapours. Throughout our experimentation and design process, we faced both setbacks and successes. Above is a crucial example of how both engineering success and failures impacted our project. While our high school did not heavily restrict access to our lab, we maximized our time when not in person by upholding weekly meetings in which we planned several forthcoming experimental designs, reviewed laboratory protocols, and developed potential educational lectures corresponding to our topic.
Figure 2: Chart demonstrating an experimental design that tests the maximum and minimum concentrations of glucose and all possible controls.
Figure 3: Data shows some successes and failures of negative controls when testing various dilutions of GOX and Glucose in 9 different trials, each containing 200 microlitres of W.R. All positive controls yielded successful results.