Difference between revisions of "Team:Oxford/Applied Design Developing"

 
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     <div style="margin-top: 100px"></div>
 
     <div style="margin-top: 100px"></div>
  
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     <h1>Applied Design<br/> <i>Developing Our Design</i></h1>
+
     <h1>Applied Design<br/> <i>Developing Our Design</i></h1><br>
  
 
<center><img class="img-responsive" width="200px;" src="https://static.igem.org/mediawiki/2017/7/7a/Applied_design_page.png"></center>
 
<center><img class="img-responsive" width="200px;" src="https://static.igem.org/mediawiki/2017/7/7a/Applied_design_page.png"></center>
  
 
      
 
      
−
     <h3>Criteria for a suitable diagnostic device: considerations from the OpenPlant Forum</h3>
+
     <h2>Criteria for a suitable diagnostic device</h2>
 +
    <h3>Considerations from the OpenPlant Forum </h3>
 
     <p>
 
     <p>
 
     Our early design criteria was influenced by research into the challenges of designing new healthcare technologies in developing countries. Our research indicated that important considerations included the level of infrastructure present, the cost to the end-user and the amount of training required.
 
     Our early design criteria was influenced by research into the challenges of designing new healthcare technologies in developing countries. Our research indicated that important considerations included the level of infrastructure present, the cost to the end-user and the amount of training required.
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<center><h6>Figure 1: Four E's framework for Applied Design</h6></center>
 
<center><h6>Figure 1: Four E's framework for Applied Design</h6></center>
  
−
     <h3>Cell-free Systems</h3>
+
     <h2>Cell-free Systems</h2>
 
     <p>
 
     <p>
 
     One of the first things we realised when planning our kit was that a cell based diagnostic would require a cold chain for transportation and maintenance of the culture once they had arrived in location and were being used for the kit. Whilst exploring the recent advantages in the synthetic biology literature we discovered a paper from Pardee at al. (2016), describing a method for freeze-drying cell lysate for use as a cell-free transcription-translation system. This was perfect, as there was no need for a cold chain, and the lysate could be produced cheaply and easily. At the open plant forum we received lots of ideas from talks by Jim Swartz and Keith Pardee. We discussed our project further with Keith Pardee, who gave us lots of practical advice on designing an optimal circuit for cell-free expression, influencing are decision to add pre-synthesised TetR to our kit rather than producing it in our kit, as we’d originally planned on doing.
 
     One of the first things we realised when planning our kit was that a cell based diagnostic would require a cold chain for transportation and maintenance of the culture once they had arrived in location and were being used for the kit. Whilst exploring the recent advantages in the synthetic biology literature we discovered a paper from Pardee at al. (2016), describing a method for freeze-drying cell lysate for use as a cell-free transcription-translation system. This was perfect, as there was no need for a cold chain, and the lysate could be produced cheaply and easily. At the open plant forum we received lots of ideas from talks by Jim Swartz and Keith Pardee. We discussed our project further with Keith Pardee, who gave us lots of practical advice on designing an optimal circuit for cell-free expression, influencing are decision to add pre-synthesised TetR to our kit rather than producing it in our kit, as we’d originally planned on doing.
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<p> <center><img class="img-responsive" width="1000px;" src="https://static.igem.org/mediawiki/2017/3/31/T--oxford--applied--design--fig3.jpg"></a><h6>Figure 2: A diagram showing the production process for cell-free technology</h6></center></p>
 
<p> <center><img class="img-responsive" width="1000px;" src="https://static.igem.org/mediawiki/2017/3/31/T--oxford--applied--design--fig3.jpg"></a><h6>Figure 2: A diagram showing the production process for cell-free technology</h6></center></p>
  
−
     <h3>Initial Design Iterations</h3>
+
     <h2>Initial Design Iterations</h2>
 
     <p>
 
     <p>
 
     Our starting point involved the detection of cruzipain in a sample of blood, however we needed to design an output system and a method for reading the output. By applying our criteria to a cell-free diagnostic system, we were able to propose some early design options. We discussed these as a group, compared the relative merits of each, and decided which would be most suitable to carry on with.
 
     Our starting point involved the detection of cruzipain in a sample of blood, however we needed to design an output system and a method for reading the output. By applying our criteria to a cell-free diagnostic system, we were able to propose some early design options. We discussed these as a group, compared the relative merits of each, and decided which would be most suitable to carry on with.
 
     </p>
 
     </p>
  
−
     <h4>Early Stage: Output Ideas</h4>
+
     <h3>Early Stage: Output Ideas</h3>
 
<h6>Table 1: Pros and Cons of initial output ideas</h6>
 
<h6>Table 1: Pros and Cons of initial output ideas</h6>
 
     <table class="table">
 
     <table class="table">
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                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>Many current spot tests use colour as an indicator</li>
+
                         <li>Equipment is easy to obtain</li>
−
                         <li>Clear presentation of the result</li>
+
                         <li>Relatively cheap</li>
 +
                        <li>Would fit into current infrastructure</li>
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>Possible to design bacteria to produce a clotting inhibitor</li>
+
                         <li>Provides a quantitative measure of coagulation</li>
−
                         <li>Can perform test on blood sample without requiring preparation</li>
+
                        <li>Decreases analysis time</li>
 +
                         <li>Can use a smaller volume of blood (fingerprick)</li>
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
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                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>Difficult to distinguish against the colour of blood</li>
+
                         <li>Involves blood collection, requiring phlebotomy skills and training</li>
−
                         <li>Can't isolate plasma easily</li>
+
                         <li>Result may not be entirely clear</li>
 +
                        <li>Inversion of the tube could affect coagulation speed</li>
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>May be difficult to visualise clotting</li>
+
                         <li>Blood clotting output might not be produced quickly enough</li>
−
                         <li>Result may be too subjective</li>
+
                        <li>Incorporating the DNA/OMV system could be difficult</li>
 +
                         <li>May not be applicable for large volumes of blood</li>
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
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     </p>
 
     </p>
  
−
     <h4>Late Stage: Ideas to measure blood clotting output</h4>
+
     <h3>Late Stage: Ideas to measure blood clotting output</h3>
 
<h6>Table 2: Potential ideas for measuring output</h6>
 
<h6>Table 2: Potential ideas for measuring output</h6>
−
    <table class="table">
+
    <table class="table">
 
         <tbody>
 
         <tbody>
 
             <thead>
 
             <thead>
 
                 <tr>
 
                 <tr>
 
                     <th></th>
 
                     <th></th>
−
                     <th>Blood Collection Tube</th>
+
                     <th>Blood collection tube</th>
 
                     <th>Microfluidics</th>
 
                     <th>Microfluidics</th>
 
                 </tr>
 
                 </tr>
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                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>Equipment is easy to obtain</li>
+
                         <li>Many current spot tests use colour as an indicator</li>
−
                         <li>Relatively cheap</li>
+
                         <li>Clear presentation of the result</li>
−
                        <li>Would fit into current infrastructure</li>
+
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
 
                 <td>
 
                 <td>
 
                     <ul>
 
                     <ul>
−
                         <li>Provides a quantitative measure of coagulation</li>
+
                         <li>Possible to design bacteria to produce a clotting inhibitor</li>
−
                         <li>Decreases analysis time</li>
+
                         <li>Can perform test on blood sample without requiring preparation</li>
−
                         <li>Can use a smaller volume of blood (fingerprick)</li>
+
                    </ul>
 +
                </td>
 +
            </tr>
 +
            <tr>
 +
                <th>Disadvantages</th>
 +
                <td>
 +
                    <ul>
 +
                         <li>Difficult to distinguish against the colour of blood</li>
 +
                        <li>Can't isolate plasma easily</li>
 +
                    </ul>
 +
                </td>
 +
                <td>
 +
                    <ul>
 +
                        <li>May be difficult to visualise clotting</li>
 +
                        <li>Result may be too subjective</li>
 
                     </ul>
 
                     </ul>
 
                 </td>
 
                 </td>
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         </tbody>
 
         </tbody>
 
     </table>
 
     </table>
 +
 
     <p></p>
 
     <p></p>
 
     <ul>
 
     <ul>
−
         <li>Blood Collection Tube: Our research showed that most blood collection tubes are lined with anticoagulation factors, in order to prevent blood from clotting. This inspired us to produce a collection tube lined with our freeze-dried cell-free system.</li>
+
         <li>Blood Collection Tube: Our research showed that most blood collection tubes are lined with anticoagulation factors, in order to prevent blood from clotting (LabCE, 2017). This inspired us to produce a collection tube lined with our freeze-dried cell-free system.</li>
 
         <li>Microfluidics: Two papers published by Steckl et al. (Lab on a Chip 2014, Biomedical Microdevices 2017) inspired us to consider a new method to screen for blood coagulation. Steckkl and colleagues presented a cheap, point-of-care blood coagulation assay, which utilised microfluidics in a paper-based device.</li>
 
         <li>Microfluidics: Two papers published by Steckl et al. (Lab on a Chip 2014, Biomedical Microdevices 2017) inspired us to consider a new method to screen for blood coagulation. Steckkl and colleagues presented a cheap, point-of-care blood coagulation assay, which utilised microfluidics in a paper-based device.</li>
 
     </ul>
 
     </ul>
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     </p>
 
     </p>
  
−
    <h3>Integrating Our Ideas Into A Design</h3>
+
  <h2 class="prototitle">Prototyping</h2>
−
     <p>
+
     <br/>
−
We began by sketching ideas on paper and sharing our thoughts during a group meeting. Following advice from Dr Tempest van Schaik, we produced a simple cardboard model of our design to view it from the end-user’s perspective. This guided some key changes for example our design originally included positive control and negative control indicators, but we realised that this created a bulky, complicated device requiring three blood samples, and would increase our costs.
+
  
−
Ultimately, we determined that by keeping the device as streamlined and inexpensive as possible, healthcare professionals using the kit could simply repeat the test for any inconclusive results.</p>
+
<img src="https://static.igem.org/mediawiki/2017/b/b0/T--oxford--prototypeimg1.png">
 +
<h3> Paper Designs and Microsafe Pipettes </h3>
 +
<div class="container">
 +
<div class="row">
 +
<div class="col-sm-8">
 +
<p>
 +
We began by sketching ideas on paper and sharing our thoughts during a group meeting. We initially based our blood coagulation test on a paper from Li, H. et al. (2014) that had described quantifying blood coagulation using chromatography membranes, however after further consideration, and helpful advice from Mike Laffan at Imperial College London we realised that if we delayed the actual ‘testing’ step of our kit, by adding sodium citrate to the reaction chamber, we could wait until we were at a binary result, rather than having varying levels of clotting that needed to be quantified by a chromatography-based method.</p>
 +
<p>This allowed us to simplify the kit, and instead use flow through a capillary tube as our test for blood clotting. The paper described above did, however, point us to an interesting product, the Microsafe Pipette. This is a very cheap volumetric pipette that allows for accurate taking of small samples of blood. As our modelling had shown us that the volume of blood was crucial if we were to produce enough bivalirudin to prevent it from clotting, we ordered some samples for testing.
 +
</p>
 +
</div>
 +
<div class="col-sm-4">
 +
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/e/e3/T--oxford--prototypeimg2.jpeg">
 +
<h6> Measuring a Microsafe Pipette </h6>
 +
</div>
 +
</div>
  
−
<h2>Current Kit</h2>
+
<h3>Cardboard Models and False Positives</h3>  
−
<p>Our current kit meets our criteria established from our 4E’s framework: it is effective, easy-to-use, economically viable and environmentally safe. A prototype version was designed using CAD software and 3D printed.</p>
+
<div class="container">
−
<br>
+
<div class="row">
−
<p> <center> <img class="img-responsive";" width="1000px" src="https://static.igem.org/mediawiki/2017/2/2c/T--oxford--kitlabelled.png"><h6>Figure 3: Annotated diagram of our kit</h6>
+
<div class="col-sm-4">
−
</center></p>
+
<div class ="imgproto">
−
<br>
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/6/6f/T--oxford--prototypeimg3.jpeg">
 +
</div>
 +
</div>
 +
<div class="col-sm-8">
 +
<p>
 +
Following advice from Dr Tempest van Schaik, we produced a simple cardboard model of our design to view it from the end-user’s perspective. The L-shape of the kit stems from our desire to prevent contamination, either from the environment into the kit or the kit into the environment. With this design the microsafe pipette can be inserted into the top of the kit and left in there when the kit is disposed of. </p>
 +
<p> We originally produced the kit with three lanes, which included positive control and negative control indicators, but we realised that this created a bulky, complicated device requiring three blood samples. This would mean more work for medical practitioners, more samples obtained from newborn babies,  and would increase our costs. Ultimately, we determined that by keeping the device as streamlined and inexpensive as possible, healthcare professionals using the kit could simply repeat the test for any inconclusive results. The incidence of blood clotting disorders in the population is so low (less than 1%), that even if these babies showed a positive result and further tests were carried out which showed a negative result, this wouldn’t be a significant issue.</p>
 +
</div>
 +
</div>
 +
</div>
  
−
<h3>Using the kit<h3>
+
<h3>Computer-Aided Design and Timestrips</h3>
 +
<p>
 +
Once we had got a ‘feel’ for what we wanted our kit to be like from our cardboard prototypes, we began to mock the kit up using Computer-Aided Design (CAD) software, this helped us to explain the system to potential stakeholders and begin to properly dimension the kit.</p>
 +
<p>
 +
Modelling of our kit had shown us that we would need to wait before checking the blood for coagulation, as bivalirudin would not be produced fast enough to prevent the clotting reaction from occurring if it had to ‘fight’ against the mechanisms of blood clotting straight away.</p>
 +
<p>We therefore designed a way in which the kit would be ‘activated’ after the optimal length of time, which from modelling we had determined to be around 10 minutes. This time was a balance between false negatives and false positives, wait less and the system hasn’t had long enough to produce enough bivalirudin to prevent clotting, wait too long and even in a situation with no cruzipain enough has been produced to stop the blood from clotting. We decided that we could design a kit which could be ‘clicked’ together after 10 minutes, where a capillary tube coated with calcium and tissue factor would pierce the compartment with the blood. The blood would then either begin to clot in the tube or not, and we could detect whether it had flowed through by the presence of a spot of blood on blotting paper at the end of the kit. We ran basic experiments with blue food colouring, capillary tubes and filter paper, and found that from a 30uL drop the food colouring would run along the capillary tube and form a dot on the filter paper.
 +
</p>
  
−
<p> <center> <img class="img-responsive";" src="https://static.igem.org/mediawiki/2017/e/e8/T--oxford--applieddesignflowchart.png"><h6>Figure 4: Diagnostic Procedure Flowchart</h6>
+
<div class="container">
−
</center></p>
+
<div class="row">
 +
<div class="imgproto">
 +
<div class="col-sm-6">
 +
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/d/d9/T--Oxford--AppliedDesign--Tubegif.gif">
 +
</div>
 +
</div>
 +
<div class="imgproto">
 +
<div class="col-sm-6">
 +
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/50/T--Oxford--AppliedDesign--blottinggif.gif">
 +
</div>
 +
</div>
 +
</div>
 +
</div>
 +
<p>
 +
Although this was a good system in theory, in practice medical practitioners would need to keep track of the progress of many kits simultaneously, and therefore it would be easy for them to misstime the kit, leading to an erroneous result. After inspiration from a cooking hood filter timer, we discovered Time-strip, and ordered some samples to test using our kit. Time-strips are cheap and easy ways to time things, just relying on the flow of a liquid through a sticker-strip. For our final device we could stick a custom timestrip on the side which would have markings for 10 and 15 minutes, and only if the liquid front is between these two should the practitioner activate the kit.
 +
</p>
  
−
<h3>Review of our kit by healthcare professionals</h3>
+
<h3>3D Printing, Professional Feedback and Final Dimensions</h3>
 +
<p>
 +
We then 3D printed some prototypes in order to actually test with the samples of microsafe pipette samples we had been sent, and in this we are indebted to Dr. Darragh Ennis of our Biochemistry Department for tireless patience in printing design after design! After refining our design, we printed a few different versions of our kit and asked medical professionals in the UK what they liked and disliked about each one.
 +
</p>
 +
<div class="container">
 +
<div class="row">
 +
<div class="imgproto">
 +
<center> <img class="img-responsive" style="height:200px" src="https://static.igem.org/mediawiki/2017/c/cb/T--Oxford--AppliedDesign--final1.gif"> </center>
 +
</div>
 +
</div>
 +
</div>
 +
<p>
 +
We spoke to a college nurse and a local general practitioner Sarah Dragonetti and Dr. Ben Riley.</p>
 +
<p>
 +
We thought they would prefer a loose, rounder hole, in order to make it easy to fit the pipette in, however they said that in fact the kits with a snugger, rounder fit made it feel more like the kit was ‘working’, and the pipettes were less likely to fall out and cause contamination. They said that a timestrip would be a good consideration for busy hospital staff, and they said that white would be the best colour because it would be easiest to see contamination with blood. One thing they mentioned that we would need to do further cost analysis on was the addition of a window to see whether the pipette had actually fully ejected the blood into the chamber. This seemed like a costly suggestion, and hopefully once a practitioner had used the kit a few times they would get used to this, but it is definitely something to run test on in a real-world setting.
 +
</p>
  
−
<p>We presented our prototype model to two healthcare professionals in order to re-evaluate our current kit. Recommendations gathered would be implemented into the future versions of our kit.</p>
+
<p>
−
 
+
After settling on our final design we asked a colleague in the engineering department, who had experience in using more sophisticated CAD software, to make our final design for us. He then produced a dimensioned drawing and final 3D file for us to 3D print.</p>
−
<h4>Mrs Sarah Dragonetti (Registered Nurse)</h4>
+
<div class="container">
−
 
+
<div class="row">
−
Findings:
+
<div class="imgproto">
−
<ul>
+
<div class="col-sm-6">
−
<li> Flat, rectangular pipette hole fits well
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/c/c8/T--oxford--prototypeimg6.jpeg">
−
<li> Timestrip would be a useful tool during busy periods
+
</div>
−
<li> Good size and good shape - feels intuitive
+
</div>
−
<li> A window would allow you to see whether the pipette was emptied, preventing someone from accidentally drawing blood back up
+
<div class="imgproto">
−
<li> A red case would make it difficult to see the blood through the window, so white or translucent casing would be better
+
<div class="col-sm-6">
−
<li> Unclear on actual device when to click together the two components
+
<img class="img-responsive" src="https://static.igem.org/mediawiki/2017/e/e7/T--Oxford--AppliedDesign--final2.gif">
−
<li> Unclear whether pipette should stay in kit or be taken out (and when)
+
</div>
−
</ul>
+
</div>
−
 
+
</div>
−
<h4>Dr Ben Riley (General Practice)</h4>
+
</div>
−
 
+
<h3>Final Kit Design</h3>
−
Findings:
+
<p>An image of this design is below. The final kit will be white, but the dimensions are correct.</p>
−
<ul>
+
<div class="imgproto">
−
<li> Kit is sealed so no worry about blood containment
+
<center><img src="https://static.igem.org/mediawiki/2017/f/f6/T--Oxford--AppliedDesign--finalkit.gif"></center>
−
<li> Ideal cost should be comparable to a one-use diabetes test strip (~£3)
+
</div>
−
<li> Size and shape is good for packaging and transport
+
−
<li> Distinctive shape will make it easy to identify
+
−
</ul>
+
−
These were very useful comments: they support some aspects of our current design, but also propose some changes which would further improve the end-user experience.
+
−
 
+
−
<h4>Overall recommendations:</h4>
+
−
<ul>
+
−
<li> Make the case transparent: prevents pipette errors but is more cost-effective than a window
+
−
<li> Make the key instructions as clear as possible, and include these within the kit
+
−
<li> Evaluate the cost of the device (which we have presented below)
+
−
</ul>
+
−
 
+
−
<h2>Integration of our kit into society</h2>
+
−
<br>
+
−
We consider integration of our device into existing healthcare systems and current infrastructure a key challenge, and therefore a fundamental aspect of our applied design considerations.
+
−
 
+
−
<h3>Dialogue with Juan Solano and Alfons Van Woerkom of The Global Fund</h3>
+
−
 
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We contacted Juan Solano and Alfons Van Woerkom of The Global Fund, an international financing organisation which has worked to fund several large-scale projects in the fight against HIV/AIDS, TB and malaria. They introduced us to the concept of market and financial analyses, in order to outline opportunities for a new diagnostic to come to market and to optimise its implementation. We reviewed a set of key points:<br>
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<li> Existing diagnostics on the market
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<li> Information on the size of the market
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<li> Regulatory framework of the country
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<li> Well documented details of the unit cost
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<li> Well documented details of the phases of production
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<li> Delivery aspects (materials, transaction costs, depreciation)
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This will allow an optimal pricing strategy to be determined at the level of the product and its users. Furthermore, it provides recommendations to potential investors regarding the product’s investment feasibility.
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  <h3> Cost </h3>
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    For costing of our kit we were aided by contact with David Sprent, an expert in International Supply Chain, and Juan Solano and Alfons Van Woerkom of the Global Fund. They helped us with costing, but also with the considerations that have to be taken into account when importing products into Latin America. We used Bolivia as a case study, and imagined what the situation would look like if the country were to adopt our kit wholesale, testing all 163,000 babies born every year. The kit would be manufactured in the UK and then transported to Bolivia, as according to <a href="https://2016.export.gov/industry/health/healthcareresourceguide/eg_main_092224.asp">Export.gov</a> as of 2014 there was no local production of pharmaceuticals.
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    <h4> Materials </h4>
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    <a href="#demo" class="btn btn-info" data-toggle="collapse"><center>Table of Costs<br>Click to expand</center>
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          <tr> <td> Bivalirudin </td> <td> 0.007 </td> <td> <a href="http://www.selleckchem.com/custom-peptide-synthesis.html">Cost (Selleckchem)</a> </br> (Modelling told us we needed 50uM) </td></tr>
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          <tr> <td> Sodim Citrate </td> <td> 1.23^10-9 </td> <td><a href="https://www.researchgate.net/post/How_much_38_citrate_do_we_need_to_add_to_anticoagulate_blood_for_platelet_rich_plasma">Amount</a> </br>
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            <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/w302600?lang=en&region=GB">Cost (Sigma Aldrich) </a>
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          <tr> <td> Calcium </td> <td> 0.00311 </td> <td><a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1538-7836.2003.00075.x/full">Amount</a> </br>
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            <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/449709?lang=en&region=GB">Cost (Sigma Aldrich)</a>
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          <tr> <td> Tissue Factor </td> <td> 8.78*10^-9 </td> <td><a href="http://onlinelibrary.wiley.com/doi/10.1046/j.1538-7836.2003.00075.x/full">Amount</a> </br>
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            <a href="http://www.abcam.com/recombinant-human-tissue-factor-protein-ab119148.html">Cost (abcam)</a>
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          <tr> <td> Capillary Tube </td> <td> 0.074 </td> <td> <a href="http://www.sigmaaldrich.com/catalog/product/aldrich/z611182?lang=en&region=GB">Cost (Sigma Aldrich)</a></td></tr>
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          <tr> <td> Injection Molding of Kit </td> <td> 0.685 </td> <td> <a href="http://www.custompartnet.com/estimate/injection-molding/?units=1">Cost (CustomPartNet)</a></td></tr>
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          <tr> <td> Cardboard Box (70x30x50mm) </td> <td> 0.1 </td> <td> <a href="https://www.abcpackaging.co.uk/">ABCPackaging</a></td></tr>
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          <tr> <td> Microsafe Pipette </td> <td> 0.15 </td> <td> <a href="http://www.safe-tecinc.com/microsafe.htm">Cost (Safe-Tec)</a></td></tr>
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          <tr> <td> Timestrip </td> <td> 0.11 </td> <td> <a href="http://timestrip.com/">Cost (Timestrip)</a></td></tr>
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          <tr> <td> Printed Instructions </td> <td> 0.021 </td> <td> <a href="">?</a></td></tr>
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          <tr> <td> TetR </td> <td> 8.25*10^-5 </td> <td> <a href="https://www.mybiosource.com/prods/Recombinant-Protein/Tetracycline-repressor-protein-class-B-from-transposon-Tn10/tetR/datasheet.php?products_id=1056262">Cost (MyBioSource)</a> </br> (Modelling told us we needed 100nM)</td></tr>
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          <tr> <td> Cell Lysate for DNA Reaction </td> <td> 0.9 </td> <td> <a href="http://www.cell.com/cell/fulltext/S0092-8674(16)31246-6?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS0092867416312466%3Fshowall%3Dtrue">Pardee et al. (2016)</a></td></tr>
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          <tr> <td> DNAse 1 Inhibitor </td> <td> 0.007 </td> <td> <a href="http://www.molcells.org/journal/view.html?year=2005&volume=19&number=1&spage=54">Choi et al. (2005) </br> Cost was assumed same as bivalirudin</a></td></tr>
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          <tr> <td> <b> Total Cost </B> </td> <td> <b> 2.06 </b> </td></tr>
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          <h4> Manufacturing Cost </h4>
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          <p> This was hard to estimate, given the unknowns in our kit, but we assumed we would ask a third-party to assemble the kit and this would lead to costs of around <b>$0.25</b> per/kit. </p>
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          <h4> Transportation Costs </h4>
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          <p> We decided that with a minimum shelf life of around a year for our test it would be pertinent to send kits once a quarter to Bolivia, otherwise we risked them expiring before being used. With a 50x30x70mm box for our kit around 8,000 can fit on a europallet after taking into account further packaging for the pallet. This means we'd be sending 5 pallets/per quarter, and we estimated that this would cost around $12,000 per shipment to get from the factory in the UK to hospitals in Bolivia. This equates to around <b>$0.30</b> per kit. </p>
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          <h4> Taxes </h4>
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          <p> Bolivia imposes a <a href"http://haiweb.org/wp-content/uploads/2015/08/Taxes-final-May2011a1.pdf"> <b>13%</b> </a> tax on pharmaceutical imports into the country.
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          <h4> <b>Total Cost</b> </h4>
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          <p>  Totalling up all these costs, and then adding 25%, as was suggested to us by those we contacted, brings the total cost of our kit to around <b>$3.90</b>, which is significantly less than the current other options on the market. </p>
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<h3>Dialogue with HeLEX</h3>
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<p> Consultation throughout with relevant stakeholders, including the Centre for Health, Law and Emerging Technologies (HeLEX) and Piers Millet, has brought some important difficulties to our attention. Key issues raised from our dialogue include: </p>
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<p>You can read more about social, economic and political factors affecting our project on our <a href="https://2017.igem.org/Team:Oxford/HP/Silver">Silver Human Practices page</a> </p>
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<p>Whilst some of these issues (e.g. dual-use) may not immediately appear applicable to a diagnostic device, biosafety and biosecurity should be considered by any groups developing new technologies using synthetic biology. A component of our Education and Public Engagement activities therefore involved approaching some of these issues in order to foster a ‘culture of responsibility’ - <a href="https://2017.igem.org/Team:Oxford/Engagement">you can read more about our activities here</a>. </p>
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<p>We discussed how optimal integration of our device partly requires established guidelines to fill in gaps which may exist in current regulation. Using Bolivia as a case-study, we have produced a policy brief which summarises some of these findings, and proposes a flowchart showing our proposed optimal diagnostics strategy for Chagas disease.</p>
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<p> <center> <a href="https://2017.igem.org/Team:Oxford/Chagas_Public_Policy"><img class="img-responsive" width="200px;" src="https://static.igem.org/mediawiki/2017/9/98/T--oxford--chagas_disease--button.png"></a></center></p>
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<p> <center><img class="img-responsive" width="500px;" src="https://static.igem.org/mediawiki/2017/e/e6/Diagnostics_flowchart.png"></a><h6>Figure 5: A flowchart showing the optimal diagnostic strategy for congenital chagas disease using a rapid protease detecting kit.</h6></center></p>
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<p>One concern raised by our dialogue with HeLEX included the importance of public engagement in addressing the awareness of Chagas disease. A public health campaign rolled out in regions of Latin America with the implementation of our kit could circumvent future issues surrounding consent and knowledge of the risks associated with Chagas. Most importantly, this would need to be translated to local languages, including Spanish, to increase access of information to local stakeholders. To this end, we have produced a draft example of a public health poster which is concise and easy to read.</p>
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<p> <center><img class="img-responsive" width="250px;" src="https://static.igem.org/mediawiki/2017/6/69/Oxford_igem_public_health.png"></a><h6>Figure 6: A draft public health poster for Chagas disease, translated into Spanish</h6></center></p>
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<h2>Future Vision for our Kit</h2>
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<p>As our kit is modular, it will be able to be easily and cheaply adapted to diagnose different diseases; the cost of changing the disease is then only the input block, not also the output block. Our vision for the future is that a streamlined manufacturing process can be established which allows a rapid development of new diagnostic modules as people characterise specific proteases which are biomarkers for disease.<p>
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<p>References:
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<h3> References </h3>
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Portable, On demand biomolecular manufacturing, Pardee
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<p>Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., … Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. http://doi.org/10.1016/j.cell.2016.04.059</p>
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The All E. coli TX-TL Toolbox 2.0: A platform for cell-free synthetic biology Garamella et al. </p>
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<p>Li, H. et al. (2014) ‘Blood coagulation screening using a paper-based microfluidic lateral flow device’, Lab Chip, 14(20), pp. 4035–4041. doi: 10.1039/C4LC00716F. </p>
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Latest revision as of 13:27, 6 December 2017

Applied Design
Developing Our Design


Criteria for a suitable diagnostic device

Considerations from the OpenPlant Forum

Our early design criteria was influenced by research into the challenges of designing new healthcare technologies in developing countries. Our research indicated that important considerations included the level of infrastructure present, the cost to the end-user and the amount of training required.

In order to gain an insight into various aspects of synthetic biology, members of our team attended the OpenPlant Forum in Cambridge, UK. Dr Tempest van Schaik gave a talk titled ’Designing Diagnostics’, using her expertise in the development of bench-to-bedside healthcare technologies and importance of the end-user experience.


From her talk, 3 key points stood out to us:

  1. Understand your analyte
    1. What is the context in which the kit will be understood?
    2. How exactly does taking blood work? Will we transport the blood to a different place, or do a spot-test by the bedside?
  2. Understand the users of the kit
    1. How will they be given the kit?
    2. Will they want to use it?
    3. Do they want it?
  3. Understand the diagnosis procedure
    1. What problems are encountered from an end-user perspective?
    2. Can we simulate a diagnostics procedure to discover any issues?


After discussing these findings as a team, we proposed a set of general criteria to guide our initial design brainstorming.

Our 4E’s Applied Design Framework

Applied design is an important component of most iGEM projects, and requires an integrated and holistic approach to ensure projects are considered from a ‘real-world’ perspective. Based on our findings from OpenPlant and research, the Oxford iGEM 2017 team came up with a framework for considering applied design - the 4 E’s (‘Effectiveness’, ‘Ease of use’, ‘Economics’ and ‘Environment & Safety’).

This provides a structured method for applied design considerations, and we hope that this framework may prove useful for future iGEM teams.

Effectiveness
  • How long will it take to get a clear result?
  • How will we ensure the test it sensitive and specific?
  • How can we ensure it can be used at the bedside?
Ease of Use
  • What equipment would be necessary to use it?
  • How can we present the result clearly?
  • What level of training would be required for the end-user?
Economics
  • What materials will be used?
  • Will it be feasible to transport our device in large quantities?
  • How can we alter it to reduce the cost?
Environment & Safety
  • Can we reduce risks associated with using GMOs in healthcare technologies?
  • How can we ensure our product is sustainable and environmentally friendly?
  • How will the end-user dispose of any used materials?
Figure 1: Four E's framework for Applied Design

Cell-free Systems

One of the first things we realised when planning our kit was that a cell based diagnostic would require a cold chain for transportation and maintenance of the culture once they had arrived in location and were being used for the kit. Whilst exploring the recent advantages in the synthetic biology literature we discovered a paper from Pardee at al. (2016), describing a method for freeze-drying cell lysate for use as a cell-free transcription-translation system. This was perfect, as there was no need for a cold chain, and the lysate could be produced cheaply and easily. At the open plant forum we received lots of ideas from talks by Jim Swartz and Keith Pardee. We discussed our project further with Keith Pardee, who gave us lots of practical advice on designing an optimal circuit for cell-free expression, influencing are decision to add pre-synthesised TetR to our kit rather than producing it in our kit, as we’d originally planned on doing.

After further reading, including papers from Garamella et. al (2016), we improved the theoretical design of our kit by discovering that we could use linear DNA from a PCR reaction for our kit, rather than plasmid DNA therby almost eliminating the risk of environmental contamination, because bacteria will not take linear DNA up as easily as a plasmid.

The final DNA-based part of the kit would be:

  • Cell lysate, which can be mass-produced
  • PCR product ofr our circuitry thtat produces the TEV protease,
  • The TetR with the specific cleavage sequence that will be produced separately and added to the reaction.

These would all be freeze-dried in the well of our kit.


During our research into cell-free, we noted a lack of information regarding its use in the field. This led us to produce a report on cell-free technology, which you can find below.



Figure 2: A diagram showing the production process for cell-free technology

Initial Design Iterations

Our starting point involved the detection of cruzipain in a sample of blood, however we needed to design an output system and a method for reading the output. By applying our criteria to a cell-free diagnostic system, we were able to propose some early design options. We discussed these as a group, compared the relative merits of each, and decided which would be most suitable to carry on with.

Early Stage: Output Ideas

Table 1: Pros and Cons of initial output ideas
Color Dye Clotting System
Advantages
  • Equipment is easy to obtain
  • Relatively cheap
  • Would fit into current infrastructure
  • Provides a quantitative measure of coagulation
  • Decreases analysis time
  • Can use a smaller volume of blood (fingerprick)
Disadvantages
  • Involves blood collection, requiring phlebotomy skills and training
  • Result may not be entirely clear
  • Inversion of the tube could affect coagulation speed
  • Blood clotting output might not be produced quickly enough
  • Incorporating the DNA/OMV system could be difficult
  • May not be applicable for large volumes of blood

From our research we found that the ‘clot-buster’ drug streptokinase is naturally produced by bacteria inspiring us to use the properties of blood to our advantage: our output could interfere with the blood clotting system to produce a result.

With a coloured dye output, visualisation of the result would require the plasma to be isolated; this would increase the complexity and cost of the test.

Furthermore, a key goal for our applied design was to ensure it could be used in a scenario with minimal training and limited resources. Findings from the National Congenital Chagas Program in Bolivia (2004-2009) showed that follow-up after diagnosis was a major difficulty in controlling the disease; as such, an immediate bedside test would be most ideal. Isolation of plasma would require resources which may not be available at the bedside/in all healthcare settings.

Having reviewed these two options, we came to the conclusion that a system based around blood clotting as an output would be most suitable.

Late Stage: Ideas to measure blood clotting output

Table 2: Potential ideas for measuring output
Blood collection tube Microfluidics
Advantages
  • Many current spot tests use colour as an indicator
  • Clear presentation of the result
  • Possible to design bacteria to produce a clotting inhibitor
  • Can perform test on blood sample without requiring preparation
Disadvantages
  • Difficult to distinguish against the colour of blood
  • Can't isolate plasma easily
  • May be difficult to visualise clotting
  • Result may be too subjective

  • Blood Collection Tube: Our research showed that most blood collection tubes are lined with anticoagulation factors, in order to prevent blood from clotting (LabCE, 2017). This inspired us to produce a collection tube lined with our freeze-dried cell-free system.
  • Microfluidics: Two papers published by Steckl et al. (Lab on a Chip 2014, Biomedical Microdevices 2017) inspired us to consider a new method to screen for blood coagulation. Steckkl and colleagues presented a cheap, point-of-care blood coagulation assay, which utilised microfluidics in a paper-based device.

Whilst simple, we decided that the blood collection tube method may not produce a clear result, which was an important consideration.

Blood-clotting System: Hirudin vs Bivalirudin

Hirudin is a 65-amino acid peptide produced by leeches, and is used widely in the medical field as an anticoagulant. It is mass produced and purified using recombinant technology; initially hirudin was proposed as the output of our DNA/OMV systems, as recombinant hirudin expression in E. coli was shown to be efficient from the literature.

Applying our 4E’s framework to our decision to use hirudin led us to explore cost-friendly alternatives. We came across bivalirudin, a congener of hirudin with a similar mechanism of action. However, importantly, bivalirudin is a smaller peptide at only 20-amino acids in length. As a result, our cost analysis showed that it would be cheaper to synthesise bivalirudin chemically than to produce hirudin recombinantly. This cost-difference provides a significant advantage in ensuring maximal availability of our kit.

Prototyping


Paper Designs and Microsafe Pipettes

We began by sketching ideas on paper and sharing our thoughts during a group meeting. We initially based our blood coagulation test on a paper from Li, H. et al. (2014) that had described quantifying blood coagulation using chromatography membranes, however after further consideration, and helpful advice from Mike Laffan at Imperial College London we realised that if we delayed the actual ‘testing’ step of our kit, by adding sodium citrate to the reaction chamber, we could wait until we were at a binary result, rather than having varying levels of clotting that needed to be quantified by a chromatography-based method.

This allowed us to simplify the kit, and instead use flow through a capillary tube as our test for blood clotting. The paper described above did, however, point us to an interesting product, the Microsafe Pipette. This is a very cheap volumetric pipette that allows for accurate taking of small samples of blood. As our modelling had shown us that the volume of blood was crucial if we were to produce enough bivalirudin to prevent it from clotting, we ordered some samples for testing.

Measuring a Microsafe Pipette

Cardboard Models and False Positives

Following advice from Dr Tempest van Schaik, we produced a simple cardboard model of our design to view it from the end-user’s perspective. The L-shape of the kit stems from our desire to prevent contamination, either from the environment into the kit or the kit into the environment. With this design the microsafe pipette can be inserted into the top of the kit and left in there when the kit is disposed of.

We originally produced the kit with three lanes, which included positive control and negative control indicators, but we realised that this created a bulky, complicated device requiring three blood samples. This would mean more work for medical practitioners, more samples obtained from newborn babies, and would increase our costs. Ultimately, we determined that by keeping the device as streamlined and inexpensive as possible, healthcare professionals using the kit could simply repeat the test for any inconclusive results. The incidence of blood clotting disorders in the population is so low (less than 1%), that even if these babies showed a positive result and further tests were carried out which showed a negative result, this wouldn’t be a significant issue.

Computer-Aided Design and Timestrips

Once we had got a ‘feel’ for what we wanted our kit to be like from our cardboard prototypes, we began to mock the kit up using Computer-Aided Design (CAD) software, this helped us to explain the system to potential stakeholders and begin to properly dimension the kit.

Modelling of our kit had shown us that we would need to wait before checking the blood for coagulation, as bivalirudin would not be produced fast enough to prevent the clotting reaction from occurring if it had to ‘fight’ against the mechanisms of blood clotting straight away.

We therefore designed a way in which the kit would be ‘activated’ after the optimal length of time, which from modelling we had determined to be around 10 minutes. This time was a balance between false negatives and false positives, wait less and the system hasn’t had long enough to produce enough bivalirudin to prevent clotting, wait too long and even in a situation with no cruzipain enough has been produced to stop the blood from clotting. We decided that we could design a kit which could be ‘clicked’ together after 10 minutes, where a capillary tube coated with calcium and tissue factor would pierce the compartment with the blood. The blood would then either begin to clot in the tube or not, and we could detect whether it had flowed through by the presence of a spot of blood on blotting paper at the end of the kit. We ran basic experiments with blue food colouring, capillary tubes and filter paper, and found that from a 30uL drop the food colouring would run along the capillary tube and form a dot on the filter paper.

Although this was a good system in theory, in practice medical practitioners would need to keep track of the progress of many kits simultaneously, and therefore it would be easy for them to misstime the kit, leading to an erroneous result. After inspiration from a cooking hood filter timer, we discovered Time-strip, and ordered some samples to test using our kit. Time-strips are cheap and easy ways to time things, just relying on the flow of a liquid through a sticker-strip. For our final device we could stick a custom timestrip on the side which would have markings for 10 and 15 minutes, and only if the liquid front is between these two should the practitioner activate the kit.

3D Printing, Professional Feedback and Final Dimensions

We then 3D printed some prototypes in order to actually test with the samples of microsafe pipette samples we had been sent, and in this we are indebted to Dr. Darragh Ennis of our Biochemistry Department for tireless patience in printing design after design! After refining our design, we printed a few different versions of our kit and asked medical professionals in the UK what they liked and disliked about each one.

We spoke to a college nurse and a local general practitioner Sarah Dragonetti and Dr. Ben Riley.

We thought they would prefer a loose, rounder hole, in order to make it easy to fit the pipette in, however they said that in fact the kits with a snugger, rounder fit made it feel more like the kit was ‘working’, and the pipettes were less likely to fall out and cause contamination. They said that a timestrip would be a good consideration for busy hospital staff, and they said that white would be the best colour because it would be easiest to see contamination with blood. One thing they mentioned that we would need to do further cost analysis on was the addition of a window to see whether the pipette had actually fully ejected the blood into the chamber. This seemed like a costly suggestion, and hopefully once a practitioner had used the kit a few times they would get used to this, but it is definitely something to run test on in a real-world setting.

After settling on our final design we asked a colleague in the engineering department, who had experience in using more sophisticated CAD software, to make our final design for us. He then produced a dimensioned drawing and final 3D file for us to 3D print.

Final Kit Design

An image of this design is below. The final kit will be white, but the dimensions are correct.

References

Pardee, K., Green, A. A., Takahashi, M. K., Braff, D., Lambert, G., Lee, J. W., … Collins, J. J. (2016). Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components. Cell, 165(5), 1255–1266. http://doi.org/10.1016/j.cell.2016.04.059

Li, H. et al. (2014) ‘Blood coagulation screening using a paper-based microfluidic lateral flow device’, Lab Chip, 14(20), pp. 4035–4041. doi: 10.1039/C4LC00716F.