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Latest revision as of 03:41, 18 October 2018
Paper-Based Microfluid Devices
Paper-based micro-fluid devices (PMD’s) can be used to help create robust, affordable, and modular diagnostics for the detection of a wide range of compounds and pathogens. The success of such tests in the detection of Ebola and Zika virus, dangerous organisms mainly affecting those in remote areas, has established PMD’s as an emerging leader in accessible diagnostic platforms. In accordance with our accessibility criteria, we thus chose a PMD as the foundation of our diagnostic assay.
At the most basic level PMD’s rely on liquid test samples flowing through predefined paths lined with dried reagents. Ideally, differences in the colorimetric output of this reaction can be used to confidently determine the presence or absence of a target compound in the sample. The functionality of a PMD is therefore reliant on process design, sample flow, reagent quantity, and reagent distribution.
Paper Based-Assay Design
The final product started with a simple early prototype. Before setting out to create this product, criteria needed to be set. This led us to create a table for our design criteria
| Customer Need | Technical Need | Technical Requirement | Target value |
|---|---|---|---|
| The test should give results fast | How fast the reaction occurs once the sample is applied | 2 minutes | 1.5 minutes |
| The test should be cheap | The test needs to cost little USD | The test will be less than 5$ | The test will be less than 2$ |
| The test should be easy to use | Test will require little steps to use | Test will have less than 4 steps | Test will have less than 3 steps |
| Test needs to be able to be shipped worldwide | Test can last several days after creation | test can last 28 days | test will last 31 days |
| test needs to be accurate | Test will very sensitive | Test will have 90% sensitivity | test will have 95% sensitivity |
Now that we know what our targets are, it is time to come up with several designs.
Generation 0
This device was an early model for an idea of a 2D design
Question 1
Should we accommodate testing sites for both vulvovaginal and invasive candidiasis on a single PMD?
Due to large phenotypic differences between the Candida albicans responsible for vulvovaginal and invasive infections, we were forced to select different biomarkers for each disease. As a consequence, a PMD testing for one type of candidiasis would require different reagents than a PMD testing for the other.
Answer: After meeting with Dr.Linnes, an assistant professor at Purdue University in the department of Biomedical Engineering, we decided to design a modular PMD which could be used to diagnose a single form of candidiasis, not both, depending on the reagents loaded into it.
Question 2
Should our assay be 2D or 3D?
Answer: Dr. Linnes indicated that a simple 3D flow through assay would be the best place to start. As it will minimize the amount of steps for the user. Her feedback was that over the counter devices with more than 3 steps tend to not get approved by the FDA. This became our first piece of HP we needed to integrate in our design.
With these basic questions answered, it was time to start getting experience in assembling and testing PMD’s. We printed a 96-well plate format on printer paper using a wax printer and tested the lanes for leaking to ensure the printing process went smoothly.
Generation 1
Question 3
How will patients know whether they test positive or negative for a yeast infection?
Answer: Initial experiments with HRP and TMB showed that the colorimetric reaction is highly sensitive, meaning small variations in reactant concentration could lead to differences in output color. This finding combined with the inability of printed color scales, as one would use with pH strips, to clearly indicate a binary ‘yes’ or ‘no’ led us to add channels for positive and negative controls to our original flowthrough model. With this modification, variations in PMD reactant concentration shouldn’t detract from a user’s ability to analyze test results
Generation 2
Question 4
Is the intermittent spacing between layers responsible for slower, inconsistent flow rates?
Answer: Yes; when paper with adhesive backing was used to assemble layers, the spacing between layers disappeared and flow rates became faster and more consistent
Question 5
Can the test accommodate viscous samples?
Answer: Yes and no, 10% glucose solution was used to emulate the viscosity of blood. Honey was used to emulate the vaginal mucus. The glucose solution flowed through the first layer, but the honey was not absorbed. Meaning if we want to test vaginal mucus, we would need to add surfactants to it.Also, it was noticed that the design had leakages during tests. misalignment in the layers allowed liquid to pass through layers outside the wax barrier.This led to the next design change to see if reducing these leakages would improve performance and consistency.
Generation 3
Question 6
Would printing wax over the entire background prevent water from leaking increase performance?
Answer: Yes, furthermore, in order to improve flow even more, the laser cut pattern on the double sided adhesive was enlarged to allow for more leniency in human error when it comes to assembling the layer.
Question 7
What order should the reagents be stored on the paper?
Answer: Testing revealed that TMB on the second layer would often react on the second layer, reducing the effectiveness of the test. So H2O2 would be placed on the second layer and TMB and our Protein construct will be placed on the last layer
Additionally, during tests it was seen that TMB would react slowly over time due to aging, so a “freshness zone” that would be visible to future customers was added. This zone would show if the TMB had reacted or not, acting as a visible shelf life marker. If the marker is yellow, then it is time to get a different test.
Collaboration and Survey
At this point, tests were sent out for the collaboration. This test was to test how well the devices would ship. 3 treatments were done. 1 was shipped wrapped with a opaque cover that could slide out and a bottom layer plastic sheet that was made to help prevent air from oxidizing the reaction. Another was shipped without the opaque cover, but with the plastic sheet. The last one was shipped without the plastic sheet on the bottom nor the cover. Before the results were received from this. We sent out a survey and received results from it. The results were very clear on a few things.
- People were concerned with not being able to tell the differences between the reaction zones of the samples
- People are concerned with possibly looking too late to read the test, this is because the TMB that doesn't react with the HRP will react with the air over time, ruining the reading.
- Not being able to tell the difference between the positive control and the sample. This is so people can actually read the results
- Buying the Test and afterwards learning it's expired.
Soon we got back the results on the collaboration. The protocol we sent to the team wasn't clear enough and they took scans of the wrong side of the paper. It was clear that although we were not able to receive the desired data, the test and directions were unclear. Thus, we changed the assay to include labels for which sides samples should be put on.
Generation 4
Question 8
Due to the confusion with the collaboration, is there a way to improve clarity on the usage of the device?
Answer: Yes, by adding a label to the top side, it might help people with usage.
Question 9
How do we allow people to differentiate the different zones as was a concern raised in the survey?
Answer: Add labels to each of the zones on the bottom layer to help people read the device
Question 10
How do we help people differentiate from the positive control?
Answer: While not having a full solution currently, some ideas are to add a device that can measure the color with the diagnostic. Another is to add a device to compare colors of known concentrations of the biomarker.
Question 11
How do we address the concern that people might by the assay and then realize its expired?
Answer: By adjusting the package in way that allows the user to look at it before purchasing it in a way that doesn’t sacrifice lifetime.
Question 12
How do we address the worries about not having enough time to read the sample?
Answer: Currently, the device will operate with a reaction within a minute. The reaction that would interfere is the creation of a yellow color on the paper. However, this only occurs if there is enough moisture or to permit the reaction. We are unsure as of now if the situation on the paper will allow for this. Regardless, this occurs no more early than 10 minutes. Due to the fact that the assay needs to be used in order for this to be an issue, it shouldn’t be too much of an issue.
Due to the survey, it was determined that people didn't want to spend more than 30 minutes on this test. Also, it was determined that we needed the device to cost less than $10. This package should come with two tests as well. After discussing things with Dr.Linnes, her experience says that in order to approved for over the counter use to approved, the device needs to be less than 3 steps. Also, the expiration needs to be about a year for the FDA to like the drug. The test also was desired to be over 90% in accuracy. These requirements are reflected in the table below with the current results of this design.
| Customer Need | Technical Need | Technical Requirement | Target value | Actual Value |
|---|---|---|---|---|
| The overall test should be fast | Time it takes for the test from sample harvesting to reading. | 30 minutes | 5 minutes | 3.5 minutes |
| The test should be cheap | The test needs to cost little USD | The test will be less than $10 | The test will be less than $2 | $0.03 |
| The test should be easy to use | Test will require little steps top use | Test will have less than 3 steps | Test will have less than 3 steps | 3 Steps, 1. Prick the blood on finger, 2. Put Blood on Sample Sites, 3. Read Results. |
| Test needs to be able to be shipped worldwide | Test can last several days after creation | test can last 1 year | test will last 1 year | TBD |
| test needs to be accurate | Test will very accuracy | Test will have 90% accuracy | test will have 95% accuracy | TBD |
Conclusion
The next step after testing was to evaluate our designs using a decision matrix. This is seen below
| Characteristics | Weight | Assay MK5 | Assay MK4 | Assay MK3 | Assay MK2 | Assay MK1 |
|---|---|---|---|---|---|---|
| Speed | 4 | 4 | 4 | 4 | 3 | 3 |
| Complexity | 4 | 5 | 4 | 4 | 4 | 5 |
| Accuracy | 5 | 4 | 4 | N/A | N/A | N/A |
| Durability | 4 | 5 | 5 | 3 | 3 | 2 |
| Cost | 3 | 2 | 3 | 4 | 4 | 4 |
| Total | 4 | 82 | 81 | 56 | 52 | 52 |
The range of values are from 1 to 5. The highest score is the most recent design. While it may not score much higher than the previous ones, it incorporates more integrated HP than the last. This means it will likely better satisfy the user's needs.
In summary, the novelness of the design presents useful opportunities for others in the field of science. Should sample testing need to be done, this hardware presents an easy and cheap way of assessing multiple samples. The user can load almost any type of reaction. They can then use the assay and scan the reaction using a photoscanner and imageJ, a free program. This presents a much cheaper alternative than using a 96 well plate with a flexx station, especially should the lab not have a flexx station. Unlike some paper-based diagnostic designs, this design differs from others due to clear labeling and fast flow and built-in filtering properties due to the different types of paper. Furthermore, this overall design can be used for other colormetric diagnostic tests. This design could revolutionize certain over the counter tests, such as pregnancy tests, std tests, glucose tests and many more in the future.
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