Design of low cost reactors for molecular diagnosis and bio engineering.

Jim Haseloff (Plant Sciences, University of Cambridge), Francesco Ciriello (Mathworks, Cambridge) and Maziyar Jalaal (Department of Applied Mathematics and Theoretical Physics, University of Cambridge).

OpenPlant Biomaker Design2020

Modern biology has exploited enzymes and clever reaction systems to produce an ever-growing range of diagnostics, environmental assays, DNA assembly and tools for education and research. Many of these molecular reactions are cheap and accessible, but require instrumentation for precise temperature regulation, programmed control over time and quantitative readout. Commercially available instruments are expensive, employing milled metal components for temperature control of hundreds of samples. There are many cases where low cost (<<£100) reactors would be very useful, even if sacrifices were made for the scale of sample numbers or flexibility or speed of temperature control.

We have taken on the challenge of exploring new open solutions for ultra-low cost micro-incubators. At the beginning of the COVID-19 lockdown, we set up an international Biomaker expert group to tackle this. Currently, around 75 participants and observers are connected via web exchange and regular teleconferencing. We have built: (i) a programmable rig for testing prototypes. The integration of no-code programming and programmable touchscreen means that the instrument can be highly flexible, and easily repurposed. (ii) This has inspired the assembly of a small control unit that can be used for low-cost implementation, after the testing process. (iii) Quantitative models for air flow and heat transfer to inform new designs and computer control. Air heating/transfer can potentially provide a cheaper and more accessible route to incubator design, compared to the conventional use of heaters attached to milled aluminium blocks. (iv) We have incorporated cheap heating hardware into custom 3D printed housings and are testing the designs.

Quantitative modelling

Figure 1: The control system for the simulated instrument (©MathWorks).

3D printing allows the design and low-cost testing of possible solutions that can include high levels of geometric complexity. One of the challenges with building 3D printed incubators is the prospect of softening or melting of thermoplastics - in our case, we'd ideally like to have a working range of zero to 95 degrees C - to encompass most commonly used DNA procedures. One of the expert group, Carlo Quinonez, has pioneered the use of 3D printing techniques to experiment with complex manifolds and flues for microscope-based cell culture incubators - and to ultimately build a high-temperature (190 degrees C) convection oven using heat resistant ULTEM 1010 plastic (https://studiofathom.com/blog/pyrahttps://www.instructables.com/id/Shape-of-things-to-come/), and we have taken inspiration from this approach.

LAMP, PCR and other incubator reaction protocols require precise and often dynamic temperature control. The thermal response of a reaction vessel, and as a result the performance of an incubator, depends on a variety of factors. These include:

•   the material properties of the device components, e.g. their thermal conductivity, density and specific heat capacity;

•   and of the fluid surrounding them, e.g. its thermal diffusivity, viscosity, density;

•   the geometry of these components, e.g. their surface areas, volumes, thicknesses, shapes, arrangements;

•   the ensuing flow patterns that result from the convection of heat along their surfaces, e.g. typified by spatially-varying flow velocities, temperature gradients and flow regimes (laminar vs turbulent flows, forced vs natural convection).

The complex nature of convective flow patterns presents many challenges to the successful design of an incubator. The correct estimation of heat transfer coefficients for components often requires detailed experimental or numerical studies. Fortunately, there is a wealth of literature when it comes to the documentation of these studies (Holman 2009) and results for simplified geometries can be drawn upon to obtain sensible estimates for these coefficients. These idealisations can inform design choices at an early stage. Relying on these, a modelling framework can be established that can successively be improved, moving forward, by a combination of hardware testing and supporting high-fidelity FEA and CFD simulations.

To build a theoretical foundation for the design, Francesco Ciriello drafted a simplified thermal model of an incubator in Simscape. Simscape is a modelling tool within the Mathworks Simulink product family that allows for the rapid design of 1D lumped-mass models for multi-domain systems.

Figure 2: Model variants were used for the controller and plants to systematically assess the impact of specific design choices on the thermal response of the samples (©MathWorks).

Lumped thermal masses are used to idealise the samples, the air in the chamber and the reaction chamber walls. Heat transfer coefficients and resistances are extracted from Holman’s book (Heat Transfer. 10th Edition, McGraw-Hill, New York, 2009) Heat Transfer book for different geometries and scenarios. The simulated instrument can now be used to experiment with design choices and controller design and tuning (Figure 1). Simulink model variants have been particularly helpful at enabling these comparisons (Figure 2).

The simulations are encouraging us to explore different parameter spaces and question traditional design paradigms, e.g. the use of resistance heaters strapped to milled aluminium blocks. Simulations are also empowering when access to hardware resources is limited. The combination of Model-Based Design, rapid prototyping with additive manufacturing technologies and low-cost electronics are a powerful enabler for collaboration on open-science projects and can democratise access to a wealth of laboratory resources for biological research and medical diagnostics.

Mazi Jalaal has used Computational Fluid Dynamics to explore important parameters, such as heat transfer coefficients, in the model explained above. He used the CDF package Comsol to simulate the fluid flow and heat transfer inside an incubator. Figure 3 shows an example of the simulations, where warm air entering the closed incubator heats a series of reaction vessels.

Figure 3: Heating eight reaction vessels inside an incubator. Colours show the temperature field

Mazi Jalaal has used Computational Fluid Dynamics to explore important parameters, such as heat transfer coefficients, in the model explained above. He used the CDF package Comsol to simulate the fluid flow and heat transfer inside an incubator. Figure 3 shows an example of the simulations, where warm air entering the closed incubator heats a series of reaction vessels.

Figure 4. Test-rig and compact control unit

Test-rig and compact control unit

One challenge was to build a test-rig that would combine the flexibility of microcontroller-based electronics with code-free programming in XOD - and speed up the development and testing of control routines and graphical user interfaces. Jim Haseloff has built a testing device with integrated programmable touchscreen to simplify design and prototyping of the instrumentation. The screen manufacturers provide software that allows drag-and-drop programming of multi-screen user interfaces, with a corresponding set of Arduino libraries. Further, XOD programmers have converted the Arduino IDE libraries to XOD libraries, with a set of nodes that allow simple visual coding of interactions between the microcontroller and screen. For example, the touchscreen can provide multiple sets of switches, knobs, gauges and level settings and sophisticated display of parameter settings or sensor measurements - all quickly reconfigurable in software. The test-rig provides a base for controlling and monitoring prototype instruments. In order to then adopt the interface for real-world instrumentation, we're experimenting with compact lower-cost hardware that can be used to build standalone instruments.

Figure 5. Components chosen for prototype, with 200µL 8-tube strip for reference.

Figure 6. 3D design for reactor vessel (left) and printed prototype with hardware installed (right).

Components for a simple prototype microreactor

Jim Haseloff selected a simple set of low-cost hardware components: PTC heater, 40mm fans, 40mm heatsinks, MOSFET modules for computer controlled switching, DS18B20 pre-calibrated digital temperature sensors and 12V DC power supply (total ~$25). These were assembled in 3D printed housings that had been designed in Autodesk Fusion 360, printed on an Ultimaker S3 printer, and were tested using the programmable test-rig assembled for this purpose.

The prototype incubator was designed for heating small (0.2 µL) reaction tubes to around 60o Celsius for LAMP diagnostics. To minimise costs and complexity, a positive temperature coefficient (PTC) heater was used as a heat source. PTC heaters provide resistive heating. When cool, the device draw a relatively large current. As the temperature rises, the resistance increases - resulting in a fall in current. This continues until the heater reaches a maximum temperature. Therefore PTC heaters do not overheat, and this simplifies the design of both the prototype control system and vessel. The prototype was intended to exploit air heating (rather than metal block heating) and vessel shape and baffles to maximise efficient heat exchange with the incubator.

Figure 7. Real-time monitoring through digital temperature sensors. Upper right panel shows the plotted output of DS18B20 temperature sensors in a prototype incubator, showing heating from room temperature (24 degrees C) to a target of 60 degrees. The heater output was controlled from XOD, and the temperature increase was logged onscreen. (The x-axis graticule marks are 20 secs apart).

Figure 8. Visualisation of heat transfer through IR sensing.

The performance of the prototype was monitored in realtime through digital temperature sensors and infrared emissions. Real time plots of DS18B20 temperature sensors in a prototype incubator show heating from room temperature to a set point of 60oC in two minutes. The microreactor could accurately maintain temperatures to within a degree at set points from 37o to 60o Celsius. This has been an encouraging start to the project, given the cost of the components, and with many avenues still to explore for improvement for efficiency, speed, cycling and real-time measurement of reactions in situ. A manufactured microreactor of this type would be cheap and find wide use for diagnostics, education and science, especially in low resource settings.

Further information: Test-rig assembly - https://www.hackster.io/jim-haseloff/programmable-test-rig-d7df62 Prototype microreactor - https://www.hackster.io/jim-haseloff/airloop-i-5d2a72 Biomaker - https://www.biomaker.org Biomaker Expert Group - contact Jim Haseloff at jh295@cam.ac.uk if you are interested in getting involved. All welcome!

Synthetic biology, coronavirus and me.

In common with most other people, I first became aware of the new coronavirus, SARS-CoV-2 (the causative agent of CoVID-19), through news reports from Wuhan in Hubei province, China in January 2020. At the time, it looked like this might be a re-run of the SARS epidemic from 2003, which was limited to China and neighbouring countries.

However, as the disease seemed to be spreading more quickly than SARS, I was asked to participate in a number of radio and TV interviews, wearing my virologist hat, starting on the 10th Feb. These early broadcasts mainly concerned a description of the virus and its symptoms and discussions of the containment measures being enforced in Wuhan and surrounding districts without much concern about the UK’s potential situation. This changed dramatically in early March when it became obvious that the disease had established itself in Italy and was rapidly moving throughout Europe.

Professor George Lomonossoff

The interviews became far more frequent and sombre in tone and started including questions about testing, social distancing, possible vaccines etc. I hope I was able to contribute useful information – it has certainly been a very interesting experience for me! I’m pleased to see that such concepts as PCR, antibody testing and R number are now in common parlance. A particular point I was able to stress was how quickly modern synthetic biology had enabled candidate vaccines to be produced, obviating the need to culture the infectious virus. However, demonstrating efficacy will take a bit longer.

Well, so much for my media work – what about a scientific contribution? Almost 20 years, I was involved in a project in the collaboration with the Pirbright Institute to develop qPCR controls to combat the 2001 outbreak of foot-and-mouth disease virus (FMDV). These were based on encapsidating FMDV sequences within cowpea mosaic virus (CPMV) particles Encapsidated mimic technology; N-Cap). This work, originally based on infectious CPMV, was successful and was applied to other veterinary diseases in collaboration the Veterinary Laboratories Agency (VLA; now called APHA).

In the past 2 years, Hadrien Peyret, a senior post-doc in my lab, has been working with Leaf Expression Systems and Meridian Biosciences to further develop the N-Cap system, using modern synthetic biology methods, as a source of PCR controls. As a result, we were in an ideal position to rapidly produce appropriate material for incorporation into qPCR-based testing kits for CoVID-19. Hadrien first discussed this on 17th March, the appropriate sequences were ordered on the 19th and arrived at JIC on the 30th.

Hadrien infiltrated plants 6th April, I harvested them on the 13th and by the 16th had purified the particles (yes folks, I really put my lab coat on and ran centrifuges!). The material is currently being evaluated by LES and St. George’s Hospital, London.

In addition to the qPCR controls my group has also been involved in expressing the structural proteins from SARS-CoV-2 in plants, with the aim of providing information to aid the development antibody tests and candidate vaccines. We were very fortunate that Jae-Wan Jung, a sabbatical visitor from S. Korea, has been working on the expression of the equivalent proteins of a related veterinary virus, porcine epidemic diarrhoea virus (PEDV). This gave us a head start and he agreed to shift the focus of his work to SARS-CoV-2.

To enable this, we applied for 12 months funding from the JIC Innovation Fund on the 30th March and received a positive response on 2nd April – not a bad turn-around and many thanks to all concerned. All the necessary DNA sequences have now arrived on-site, so it’s full steam ahead with expression studies.

That’s about it as of 1st May – stay safe.

Written by OpenPlan PI Professor George Lomonossoff

Bio-foundries in the Age of Pandemics

Biofoundries are integrated platforms for the rapid design, construction, and testing of genetically reprogrammed organisms for biotechnology applications and research. In practice, their application to biotechnology and genetic engineering lies mainly in the expertise of the scientists that work within them. Physically, Biofoundries are suites of laboratory automation equipment that could, conceivably, be applied to numerous scientific workflows. For example, the Earlham Biofoundy has five main automated platforms: an automated cryostorage system that will deliver a 96 well plate populated with a user-specified array of any of the 80K tubes in its store; a liquid handling system that moves nanolitre droplets of reagents (or cells) using acoustic energy; two highly flexible multi-station micro-scale liquid handling systems, and an automated microfermentation platform for optimising, monitoring and sampling microbial cultures. Similar equipment can be found in labs that conduct high-throughput genotyping, or in pathology labs, including diagnostic facilities.

Automation Platforms at the Earlham BioFoundry

Last year, the Global Biofoundry Alliance (GBA) was established to coordinate activities across the Biofoundries that are popping up across the world. While there are similarities in the goals of Foundries and, to an extent, their facilities, the projects and expertise varies between Foundries. Across the Biofoundries a wide array of molecular biology and biochemical workflows have been miniaturised, scaled and automated. In addition, numerous software packages have been developed to aid the design and analysis of experiments.

The Logo and Objectives of the Global Biofoundry Alliance

Along with specialised workflows for specific projects, most Biofoundries have miniaturised and automated at least one type of parallel DNA assembly reaction (e.g. Golden Gate and Gibson Assembly) together with several other core molecular biology workflows such as DNA/RNA purification, PCR set-up etc. All Foundries are able to perform at least hundreds of such reactions each week, and some have projects that require throughputs of thousands, or even tens of thousands.

As SARS-CoV-2, the virus strain that causes COVID-19, began to spread across the globe, it became clear that the pathology labs in many countries did not have the necessary capacity to meet demand. Further, it became apparent that the global demands for reagents required for testing, particularly for specific brands of reagents, were outstripping supply.  While a few Biofoundries with existing expertise or projects in diagnostics and infectious diseases have continued to work, unfortunately most Biofoundries have been shut down as they are not certified as diagnostics laboratories, and thus many automation platforms are sitting unused as Biofoundry staff moved to working from home.

However, the scientific community has responded astoundingly quickly with several inventive detection protocols being developed and published. It has also responded to support the infrastructures already approved for diagnostics. In Norwich, movable liquid-handling platforms were relocated from the Earlham Institute to the Norfolk and Norwich University Hospital to enable automation of the approved RNA-extraction protocol. At the time of writing, additional automation from the Earlham Biofoundry is being scoped for deactivation of virus on swabs and the preparation of reagents for the approved qPCR tests. The team at the London DNA Foundry have also relocated several automation platforms to their local hospitals to increase capacity in the capital. These automation platforms also require the presence of automation specialists to program and validate the workflows and to train new operators.

As pleased as we are to offer these small measures of assistance, there is a degree of frustration that the Biofoundries, filled with appropriate equipment for high throughput testing, are not enabled to do more. Interestingly, one of the exercises planned for the next meeting of the Global BioFoundries Alliance (previously planned for May 2020 in Canada), was to agree a series of activities that Biofoundries could undertake to address a Global Challenge such as emerging diseases. Sub-groups working on software, metrology and the development of the challenge were formed last year and had already began to formulate ideas. 

Since the pandemic has taken hold, we have continued these discussions with the main question being "Can Biofoundries develop generalised workflows for biosecurity threats, and, if so, what needs to be done?"

Interestingly, the many discussions all seemed to boil back down to the same aims that were initially described as the core business of the GBA:

·        To test the robustness of protocols across different platforms and locations

·        To establish infrastructures and a community of practice that would enable the rapid transfer and benchmarking of protocols on new platforms and in new locations

·        To establish and scale-up low-cost protocols, including those that can be performed on low-cost platforms

To these, Covid-19 has added:

·        The need to validate protocols using locally-manufactured reagents, which obviously includes the ability to make and validate the quality of key reagents such as reverse-transcriptases

·        A greater focus on the development of workflows on deployable automation platforms that can be relocated to the point-of-of need (e.g. approved diagnostic laboratories)

It is still unclear how much these current efforts will impact the current pandemic. But, this pandemic is highlighting some of the weak links in biosecurity and preparedness, showing us where we can and should focus our efforts to be able to deal with future challenges.

Written by Dr Nicola Patron, OpenPlant project leader.

Iceni Dianostics, an OpenPlant spinoff, contributes to the fight against COVID19.

In December 2019 the World Health Organisation received the first reports of a previously unknown virus, Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2).

Professor Rob Field, OpenPlant PI and Iceni Diagnostics cofounder.

Coronaviruses are a large group of viruses that are known to infect both humans and animals and cause a range of respiratory illnesses. The main symptoms of the virus outbreak, known as COVID19, include a fever and a continuous cough but in severe cases the disease can cause pneumonia and bronchitis which can cause death in some cases. 

The COVID19 outbreak has seemingly brought the world to its knees with many countries enforcing lockdown measures to reduce transmission rates and with over 32,000 deaths reported in the UK alone.

The scientific community has been fast to react to the novel outbreak, with the virus genome being discovered and published within the first four weeks of its detection. Now scientists across the globe are working to develop vaccines and diagnostic tools that help detect the virus.

With vaccine production and mass manufacture being lengthy and costly, many scientists are turning to developing diagnostic tests that can help in the bid to slow the transmission rate and spread of the disease at the point of care.

Many of the current COVID19 tests are largely based on PCR (polymerase chain reaction) that requires laboratory setting for analysis and knowledge of the virus’s genetic code.

However, Iceni Diagnostics, an OpenPlant spin off company based in Norwich are developing a new approach that identifies the virus using sugar (glycon) recognition instead of the viral genetic code, which can mutate.

Iceni Diagnostics existing prototype for influenza can detect the virus in less than 20 minutes, OpenPlant PI Professor Rob Field and his team are now working on adapting this test to detect the COVID19 virus.

The innovative handheld device requires a sample of saliva and uses lateral flow to give a simple positive or negative result. An important design feature is that it will require no training to operate allowing it to be used in any location by any individual.

To find out more about Iceni Diagnostics and their efforts to combat COVID19 visit their website here.

Digital Workshop: No-Code Programming for Biology

Sadly, in order to protect our community, the popular No-Code Programming for Biology workshop due to be held on 23rd-24th March was cancelled. However, we’re pleased to announce that the materials for this workshop are now being made available online.

This course was designed to introduce biologists to the basics of building custom instrumentation to assist with experiments in the lab and field. Part of the long-running Biomaker project, the course covers the fundamentals of using Arduino-based microcontrollers, sensor electronics, displays and actuators, as well as use of the visual programming language XOD. By combining these skills participants can learn to build a variety of instruments that are useful for measuring and controlling biological systems.

Making your own devices for use in biological research can be advantageous in many ways – components are often very cheap compared to commercially available options; there is a well-established community of experienced scientists focused on collaboration and open sharing of designs; and the opportunities for customisation and experiment-specific adjustments are endless. However, there are also hurdles for those looking to build custom instruments. Without a background in electronics or engineering it can be hard to know where to start. In addition, many systems require the maker to be able to understand and write code in languages such as CC+, which take time to learn and contain complex syntax.

Biomaker and the No-Code Programming for Biology workshop aim to break down some of these barriers. The course provides biologists with a hardware starter kit, a series of simple tutorials to get started using hardware and electronics, and an introduction to the visual programming language XOD. Instead of having to write code to control your instruments, XOD uses ‘nodes’ to represent different hardware and functions. Connecting these nodes in different combinations allows you to control your hardware and customise your instrumentation.

Previously, teams working on Biomaker projects have used these concepts for a wide variety of applications, including instrumentation, microscopy, microfluidics, 3D printing, biomedical devices, DNA design, plant sciences and outreach and public engagement. The No-Code programming for Biology workshop builds on what we have learned from running Biomaker projects and provides biologists with the necessary skills to start building their own devices and advance their research with inexpensive custom instrumentation.

If you would like to get involved with the No-Code Programming for Biology course the introduction, and first set of tutorials are now available on the Biomaker website. A number of hardware starter kits may be available upon request, although this cannot be guaranteed.

More information about the Biomaker project can be found on the Biomaker website. For questions and enquires please contact the Cambridge SynBio IRC and OpenPlant Coordinator Steph Norwood at coordinator@synbio.cam.ac.uk.