Design of air flow based microreactors
Temperature-controlled incubation of enzyme reactions are a key part of any molecular biology workflow. These might range from simple digestions at 25ºC to 37ºC, but the advent of polymerase chain reaction (PCR) for DNA amplification in the 1909's triggered the development of a range of more capable instruments that could rapidly and accurately through higher temperatures 50ºC to 95ºC. Sophisticated devices with Peltier devices and electronic feedback for temperature control have become routine fixtures in modern labs. However, these can be relatively expensive for researchers and educators/students in low resource settings.
One early strategy for temperature control exploited the use of circulating hot and cool air to incubate PCR reactions. For example, hand-built prototypes used hair dryer and vacuum cleaner parts for controlled air flow (see below). This approach led to a more refined commercial instruments - including the Roche LightCycler (below) and the Corbett RotorGene, some of the first PCR instruments with real-time fluorescence monitoring of DNA amplification.
Instruments based on air flow heat transfer have largely been supplanted by direct contact with machined metal blocks and Peltier based heating/cooling. However, the wide availability of 3D printers, high temperature-resistant filaments and cheap electronic controllers suggested that these early ideas might be worth revisiting for the design of modern low cost devices.
“For a real-time system, the light bulb in the thermal cycler had to be replaced with another source of heat. We went back to the hair dryer concept. The dryer and vacuum cleaner were connected to the center drum by vacuum hose tubings. This contraption was so loud! It was remarkable that it worked as a thermal cycler.”
Carl Wittwer Lab, University of Utah https://arup.utah.edu/media/rapid_pcr/rapid_pcr_slides.pdf
Early trials
In 2020, the onset of the COVID pandemic triggered great interest in accessible diagnostics. One of the challenges was to find ways of building instruments for running temperature-controlled LAMP and PCR reactions in low resource environments. Such instruments would have wide applications in other areas of molecular engineering too. Starting from a self-admitted naive position, I explored a series of investigations, starting with simple vessels with heater and recirculating air through to experiments with different geometries, types of fan, heat sinks, manifolds, 3D printed materials and microreactor features. These have been documented in a series of technical posts on Hackster.io, linked below. The exploration and testing of various in design strategies and use of different off-the-shelf components could be of interest to other builders...
Summary of Airflow Reactor development: Testing construction principles for building open source, low-cost microreactors for biological diagnostics and DNA engineering.
AirLoop I (Part 2): Attempt to build an ultra-low cost molecular reactor, using computer controlled recirculating air.
Radial Airflow Incubator (Part 3): Design of a low cost incubator with heated radial airflow for molecular diagnostics.
Heatsinks for Microreactor Temperature Control (Part 4): Testing different heatsinks for temperature control by forced air convection in low cost microreactors.
Modular Microreactors (Part 5): Testing designs for low-cost microreactors for biological diagnostics and DNA engineering.
Microreactor heating and airflow circuits (Part 6): Design and testing of alternative air heating and recirculation systems for low-cost instruments for molecular diagnosis and DNA engineering.
Thermal Cycling Airflow Reactor I (Part 7): Part of a challenge to build low-cost microreactors for DNA engineering, this project explores switched airflow to build a thermal cycler.
Thermal Cycling Airflow Reactor II (Part 8): Adding an airflow manifold to a very low cost microreactor, designed for DNA assembly, PCR and programmable biology.
Refactoring the Airflow Reactor design (Part 9): Redesigning the Airflow reactor for final testing and open application development.
Airflow reactor manifolds (Part 10): Testing 3D printed manifolds for the AIrflow Reactor: part of a project series to build low cost devices for DNA engineering and diagnostics
High static pressure fans for the Airflow Reactor (Part 11): Testing upgraded fans for better forced convection and improved heat transfer in 3D printed reactor vessels.
Redesigning flow channels for the airflow reactor: Improvements in switched air flow for a low cost device for DNA engineering and diagnostics.
The experiments were consolidated in the design of an isothermal incubator that was built from heat resistant 3D printing filament, 50W car window heater, computer tangential fan with electronic sensor, XOD software control and display, and documented here on Hackster:
AirFlow microreactor: Working low cost device for constant temperature incubation of micro reactions, including LAMP molecular diagnostics.
The construction of the device was also documented in the Biomaker handbook for Rapid Prototyping, and the relevant extract can be downloaded here as a PDF. The instrument is relatively low cost, even cheaper if one economises with the display, is temperature-accurate and works well for running LAMP diagnostic reactions, for example. The design has provided a springboard for further work to include heating and cooling cycles, and to develop a cheap airflow instrument capable of PCR amplification of DNA, and other reactions that require higher temperatures or cycling.
Building the AirFlow isotherm microreactor
Earlier prototyping experiments (see Background section below) have led to the current design of a programmable thermal reactor that allows microtube reactions to be incubated in a constant temperature air flow. The objective was to build a device that included:
Space for at least one 8-microtube strip that takes up a volume of approximately 80x30x10mm - with allowance for possible future integration of optical sensors or cheap plastic fibre optics.
An off-the-shelf PTC resistive element as heat source.
Unidirectional fan-forced air flow through the device, using a low-cost computer blower fan.
Minimal volume of air for recirculation, to improve response to heating (or cooling).
An accurate thermal control system using low-cost Arduino electronics and sensors.
Touchscreen microcontroller interface that allowed easy use of the programmable device.
Attempt to minimise costs of construction and use components that are globally accessible.
The use of 3D printing, no-code programming, commodity electronics and open source documentation to allow free sharing and modifications of the design.
The project aimed to explore different components, designs and practical assembly of low-cost microtube incubators - which would make accessible a new generation of isothermal reactions - for home testing, field applications and international educational efforts.
Instrument design
The core of the instrument is a rack for an 8-microtube strip inside a modular 3D printed set of blocks that create a physical loop for air flow - with temperature control by a computer controlled heater and forced air flow by a blower fan.
The device consists of sections that slot together with half-lap joints defined in the 3D print files. Exterior walls are 8mm thick, with 3.75 mm flanges at the overlap between section, allowing 0.5 mm gap between half-lap joint pieces, which are 5mm deep. This allows sufficient clearance to avoid problems with fitting of curved pieces, and allows the sections to be simply stacked on top of each other to create a fairly stable and air tight joint. This is very useful during prototyping, but might be replaced by something more permanent in a finalised design.
The sections consist of (i) a base plate with an extended front deck for the electronics, (ii) a mid-vessel sections sits above this and contains the heater and fan, and support for the microtube rack. (iii) A custom lid sits above this, and contains streamlined venting to direct forced air flow between the fan and heater compartments. (iv) A front console sits on the front part of the base section, This provides a support for the integrated touchscreen and a housing for the instrument electronics.
The AirFlow parts can be printed in a variety of different materials, but care must be taken if the reactor is to be used at temperatures over 70ºC, where commonly used materials such as PLA will start to soften and deform, and may even melt. I have settled on Extruder GreenTEC Pro filament, which is relatively resistant to heat up to 160ºC (VICAT softening temperature: at which a specimen is penetrated to a depth of 1 mm by a flat-ended needle with a 1 mm² circular or square cross-section - but I try not to stray too much above 100ºC). GreenTEC Pro is also derived from renewable raw materials and is biodegradable. The material is very easy to print with and produced a matt finish which is easy to work with tools or smooth with abrasive pads after printing.
The components are relatively large and can take over a day to print. Print failures were minimised by the use of a print bed adhesive. I have found that Dimafix is an excellent adhesive for use with GreenTEC Pro filament on the heated glass bed found on the Ultimaker S3. The build is released after printing by cooling or refrigerating the plate. Printing is generally trouble-free. Also, GreenTEC Pro Carbon is available for stronger carbon-fibre-infused assemblies, using a CC Red 0.6 print core with the Ultimaker S3.
biomaker.org