This page provides a summary of our results and will be divided into 2 major sections: extraction and concentration. We will provide a comparison between using Chyritect and lab methods and highlight key advantages of our device. Finally, we will list the next few steps we intend to take to integrate the device and make it a fully-functional, independent diagnostic device. After discussions with various stakeholders, we focused our efforts on reducing the turnaround time while increasing the device sensitivity as much as possible.
Our first set of experiments were to test the extraction of the inoculated Bd and Bsal plasmids DNA from E.Coli using the Surface chemistry method that we developed. We then compared these values with the concentration of DNA extracted using the standard QIAprep Spin Miniprep Kit. The following are the results obtained:
Table 1: DNA Extraction Results from QIAprep Spin Miniprep Kit protocol
Table 2: DNA Extraction Results from Prototype Microfluidic Chip
The average concentration from the QIAprep Spin Miniprep Kit protocol was observed to be 116.6 ng/µl. The average concentration from the Prototype Microfluidic Chip was observed to be 7.2 ng/µl. While both process took approximately the same amount of time (30 mins), it is worth noting that the QIAprep spin Miniprep kit involves numerous steps with lab components like a centrifuge and multiple different buffers while the extraction microfluidic chip minimizes human intervention greatly. While the difference in the amount of DNA extracted was significantly lower in the microfluidic component, we have since come to realize that it is still sufficient for downstream detection using the electrokinetic concentration method.
We were determined to increase the extraction efficiency so after a deep dive into existing literature we found that DMP (dimethyl pimelimidate) can be used as a linker in the DNA solution to strengthen the ability of the target DNA to cling to the inner walls of the microfluidic chip. Additionally, we learnt that Tris EDTA (TE) Buffer can be used to dissolve the DNA that clings to the walls in the final extraction step.
The following tables show the subsequent experiments we designed to increase the efficiency of extraction of DNA.
Table 1: The represented data of the first trial of checking the efficiency of the DNA extraction through Nanodrop2000.
Figure 1: Graph representing the extraction efficiency rate according to the time of incubation.
Table 2: The represented data of the first trial of checking the efficiency of the DNA extraction after the addition of DMP linker through Nanodrop2000.
Figure 2: Graph representing the extraction efficiency rate according to the time of incubation (TE Buffer/DMP Linker).
Figure 3: Graph representing the difference in between the extraction efficiency depending on the addition of TE Buffer/DMP Linker, according to the time of incubation.
Therefore we were able to optimize our extraction methods to increase efficiency and reduce the time taken to 30 mins after which the DNA solution is passed on to the concentrator chip for detection. In this process we also adopted the Engineering Design Cycle (Design → Build → Test → Learn→ Design...) to help us create a framework to work with.
We envision the extracted DNA to be passed into the concentrator chip where it will use electrokinetic concentration to focus the target DNA into a small region where it can interact with CRISPR Cas12a molecules and produce a fluorescent signal. In this set of experiments, we first tested the chip with a high concentration of Bd (136.5 ng/ul) and recorded the fluorescent signal and compared it with a control sample. The 2 graphs below show the results of these tests.
Figure 5: A visual representation of the fluorescence of a positive sample compared to the control test.
In both of the above graphs there is a clear increase in the fluorescence intensity after 200 seconds of adding CRISPR reagents. In a lab setting this result takes 35 minutes to confirm the presence of Bd using CRISPR reagents. This could be attributed to the sensitivity of the optical microscope used in the lab once the DNA is concentrated but we envision our device to use photoelectronics to detect a change in fluorescence intensity.
Next, we decided to check the device's limit of detection and compare that to lab values for the same. The Biology team found the limit of detection in a lab setting to be 103 copies/ul (Figure 5). We used 50 ul aliquots of the same sample and ran tests on the concentrator chip and measured fluorescence intensity as a function of time and compared the values to the control sample. (Figure 6).
The above results clearly indicate an increase in fluorescence intensity for all samples as low as 1 copy/ul. It was however not possible to make a quantifiable model for increase in fluorescence intensity as a function of concentration of target DNA as there was a significant amount of fluctuation between the different concentrations. We didn’t further explore this as Dr. Penner had mentioned that he did not need quantitative data. To make more reliable conclusions, we intend to repeat the above tests and compare the results.This test however shows the limit of detection to be as low as 1 copy/ul which makes electrokinetic concentration 1000 times more sensitive than present lab techniques.
To complete the device, we intend to use photoelectronics to sense the subtle changes in emitted fluorescence intensity however the final module is still being constructed. In the meantime we intend to use a portable microscope along with the concentrator chip and the extraction module to accurately detect for the presence of Bd and Bsal in a sample.
