Team:Oxford/Applied Design Current
Applied Design
Introduction
iGEM encourages all teams to take their projects beyond the lab and to take a holistic approach to design. The question “What is our real world problem?” has been a key consideration from the beginning, and has guided our project throughout the summer.
To put our diagnostic device into context, we considered various aspects including safety, accessibility and socioeconomic factors in Latin America. Many design iterations were built upon over the course of the summer, influenced by discussions with experts from a range of disciplines, including blood coagulation, microfluidics and general diagnostic devices.
Having thoroughly examined and evaluated various design options, we propose a final design for our system which fulfills our criteria for a suitable diagnostic device.
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:
- Understand your analyte
- What is the context in which the kit will be understood?
- How exactly does taking blood work? Will we transport the blood to a different place, or do a spot-test by the bedside?
- Understand the users of the kit
- How will they be given the kit?
- Will they want to use it?
- Do they want it?
- Understand the diagnosis procedure
- What problems are encountered from an end-user perspective?
- 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.
- 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?
- 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?
- 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?
- 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 |
|
|
| Disadvantages |
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|
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 |
|
|
- 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.
- 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.
Integrating Our Ideas Into A Design
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.
Current Kit
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.
Figure 3: Annotated diagram of our kit
Using the kit
Figure 4: Diagnostic Procedure Flowchart
Review of our kit by healthcare professionals
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.
Mrs Sarah Dragonetti (Registered Nurse)
Findings:- Flat, rectangular pipette hole fits well
- Timestrip would be a useful tool during busy periods
- Good size and good shape - feels intuitive
- A window would allow you to see whether the pipette was emptied, preventing someone from accidentally drawing blood back up
- A red case would make it difficult to see the blood through the window, so white or translucent casing would be better
- Unclear on actual device when to click together the two components
- Unclear whether pipette should stay in kit or be taken out (and when)
Dr Ben Riley (General Practice)
Findings:- Kit is sealed so no worry about blood containment
- Ideal cost should be comparable to a one-use diabetes test strip (~£3)
- Size and shape is good for packaging and transport
- Distinctive shape will make it easy to identify
Overall recommendations:
- Make the case transparent: prevents pipette errors but is more cost-effective than a window
- Make the key instructions as clear as possible, and include these within the kit
- Evaluate the cost of the device (which we have presented below)
Integration of our kit into society
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.
Dialogue with Juan Solano and Alfons Van Woerkom of The Global Fund
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:- Existing diagnostics on the market
- Information on the size of the market
- Regulatory framework of the country
- Well documented details of the unit cost
- Well documented details of the phases of production
- Delivery aspects (materials, transaction costs, depreciation)
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.
Cost
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 Export.gov as of 2014 there was no local production of pharmaceuticals.Materials
Click to expand
| Component | Cost Per Kit ($) | Source |
|---|---|---|
| Bivalirudin | 0.007 | Cost (Selleckchem) (Modelling told us we needed 50uM) |
| Sodim Citrate | 1.23^10-9 | Amount Cost (Sigma Aldrich) |
| Calcium | 0.00311 | Amount Cost (Sigma Aldrich) |
| Tissue Factor | 8.78*10^-9 | Amount Cost (abcam) |
| Capillary Tube | 0.074 | Cost (Sigma Aldrich) |
| Injection Molding of Kit | 0.685 | Cost (CustomPartNet) |
| Cardboard Box (70x30x50mm) | 0.1 | ABCPackaging |
| Microsafe Pipette | 0.15 | Cost (Safe-Tec) |
| Timestrip | 0.11 | Cost (Timestrip) |
| Printed Instructions | 0.021 | ? |
| TetR | 8.25*10^-5 | Cost (MyBioSource) (Modelling told us we needed 100nM) |
| Cell Lysate for DNA Reaction | 0.9 | Pardee et al. (2016) |
| DNAse 1 Inhibitor | 0.007 | Choi et al. (2005) Cost was assumed same as bivalirudin |
| Total Cost | 2.06 |
Manufacturing Cost
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 $0.25 per/kit.
Transportation Costs
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 $0.30 per kit.
Taxes
Bolivia imposes a 13% tax on pharmaceutical imports into the country.
Total Cost
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 $3.90, which is significantly less than the current other options on the market.
Dialogue with HeLEX
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:
- Dual-use technology in synthetic biology
- Management of data gathered from our device
- Transnational boundaries and international collaboration
You can read more about social, economic and political factors affecting our project on our Silver Human Practices page
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’ - you can read more about our activities here.
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.
Figure 5: A flowchart showing the optimal diagnostic strategy for congenital chagas disease using a rapid protease detecting kit.
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.
Figure 6: A draft public health poster for Chagas disease, translated into Spanish
Future Vision for our Kit
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.
References: Portable, On demand biomolecular manufacturing, Pardee The All E. coli TX-TL Toolbox 2.0: A platform for cell-free synthetic biology Garamella et al.
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