Dual-Syringe Continuous Pumping Mechanism
The DSCPM: Combining Existing Ideas
The Dual-Syringe Continuous Pumping Mechanism (DSCPM), APUS’s pumping mechanism combines the concepts of existing pumping mechanisms with the idea of rectification in electronics. Instead of having one syringe that needs to be refilled every time it depletes, with the DSCPM, we have two syringes that sweep inversely to one another- that is, while one infuses, the other refills, and visa versa. Any work done by the syringes is then rectified into forward flow via solenoid operated valves. This is the fluidic equivalent of bridge-rectifying AC electric current into DC current (the only conceptual discrepancy being that AC electric current is sinusoidal, while the “AC flow” generated with our syringes is square wave).
Having two syringes generate square wave “AC flow” should theoretically give us flawless forward flow when rectified. Since the syringe does not need to be replaced at the end of a sweep like in traditional syringe pumps, it doesn’t matter how often a sweep is completed. This allows us to use very small syringes and vastly decrease the precision needed in terms of linear actuation (aka. the motor and gear mechanism can be very inexpensive, and we can achieve the same laminarity as traditional syringe pumps).
Comparing The DSCPM with Existing Products: Specifications
According to the published specifications of the Harvard Apparatus PhD 2000/ULTRA Syringe Pumps, continuous (laminar) flow is achieved when a minimum increment of flow generation is occurs at least once every 27.3 seconds. This metric was obtained via “reverse engineering” Harvard Apparatus’ calculations from their specification, and the metric of about 30 seconds per minimum increment was confirmed in our Human Practices meeting with Harvard Apparatus. Considering this, the calculated specifications of the DSCPM are as follows:
As is demonstrated in the above figure, when configured with a High-Torque Servo Motor and the current gear mechanism, the DSCPM can small flow rates while maintaining laminarity.The flow resolution of the DSCPM may not be as small as the HA PhD 2000, but there are some benefits to the DSCPM, the first of which being the cost. The total cost of the DSCPM is $500, which is significantly less expensive than similar lab equipment. Additionally, the DSCPM can generate continuous flow via flow rectification by the solenoid pinch valves. Harvard Apparatus and other companies, such as New Era Pumps, sell “continuous flow kits” that are usually kits with dual check valves that passively rectify flow. While check valves rectify almost flawlessly at high flow rates, they damage laminarity severely at very small flow rates (on the order of µL/min) due to the minimum “cracking pressure” required to open them in the forward direction. For microfluidic research that requires small flow rates, the DSCPM provides more stable laminarity. The specifications for the DSCPM include the minimum laminar flow rates with different volume syringes. In APUS, we use 10µL syringes.
Comparing The DSCPM with Existing Products: Performance Metrics
The Pressure vs. Time plots for the DSCPM and the Harvard Apparatus PhD 2000 (the control and “golden standard”) are below. Data was collected with a Honeywell ABP2 Series differential pressure sensor that was placed in series with the DSCPM and the waste.
Although the laminarity of the DSCPM is lower resolution than the resolution of the PhD 2000, it has been shown that the disruptions in the laminarity do not have adverse affects on the cells. The GIFs below show cells in their monolayer chambers during one of the pressure spikes visible in the Pressure vs. Time plots. It is visible that the cells are not disturbed at all.
Microfluidic Motherboard
The concept of having a motherboard was introduced from the idea of an electric motherboard serving as a control to allow connection through multiple components in a system. In a similar way, the APUS Motherboard is adapted to direct flow coming from the DSCPM to the PDMS chips that have cells housed in them. When designing the Motherboard for APUS, we wanted it to be customized to perform various functions as highlighted in Motherboard Operations Diagram below.
Moreover, the Motherboard is “plug-and-playable” and automated, making APUS adaptable to multiple pathways of fluidic flow, allowing experiment customization without human intervention.
The most crucial aspect of the Motherboard is its ability to control flow and facilitate plug-and-play functionality. The motherboard is composed of five essential layers: control, adhesive, silicon, adhesive, and flow layers. It operates through a vacuum-air solenoid system, where solenoids create either a vacuum or allow air to flow into connected valves. The silicone layer moves up and down at these valves to regulate the passage of fluids within the channels. The diagram below effectively illustrates how applying vacuum or air to the valves in the control layer influences the flow direction.
The Motherboard's solenoid controls are managed through a GUI software, enabling users to customize experiments by predefining the sequence in which solenoids activate and deactivate. Additionally, the software permits users to select the number of PDMS chips (strains/microbes) they wish to include in the experiment. APUS operates in an automated manner, allowing users to preconfigure experiments using the software. Users can instruct it to change media/pathways at specified times and then leave it unsupervised. APUS autonomously directs the flow according to the preset operations and durations, providing a convenient and efficient approach to experimental control.
Motherboard Operations Menu
Along with the Motherboard Operations previously mentioned, below are examples of the specific pathways users can customize, demonstrating how the fluid travels throughout the Motherboard depending on which valves are open or closed.
Environmental Control Chamber
The temperature-controlled chamber is a major breakthrough, carefully designed for efficient cell growth. It's great for maintaining precise temperatures, crucial for the best results. The chamber is flexible, fitting different microscopes and ensuring stable temperatures while observing cells. The main aim is to give users control over the temperature that suits their specific cell types.
Hardware design
The design for the cell temperature chamber was crafted to seamlessly accommodate various microscopes and securely house cells while maintaining a precise temperature using PID temperature control technology. This adaptable feature is strategically placed below the microscope, enabling seamless data recording without disturbing the controlled environment or removing cells. It is adaptable to any microscope since we offer the STL file where predefined parameters can be changed to fit the prefered microscope. This flexibility ensures that the chamber integrates seamlessly with different microscope configurations. Through rigorous prototyping and testing, the chamber was successfully connected to a heater using a thermally insulated tube, optimizing heat transfer and enhancing overall hardware efficiency. Additionally, a clever solution of incorporating direct airflow through a vent was implemented to attain the desired temperature within the cell chamber. Thanks to the well engineered hardware the environmental control chamber is to maintain accurate and consistent heat levels without causing overheating in the heater holder. Moreover, this design offers researchers a reliable and effective tool for their cellular studies, striking a balance between adaptability, efficiency, and precise environmental control.
Avoid temperature spikes by predicting future temperature
The PID controller designed, tailored for a relay-based system, demonstrated remarkable capabilities, enabling analysis of real-time sensor data in relation to the desired setpoint. The controller effectively evaluated current, past, and future values to generate precise outputs that helped maintain temperature stability within the system. In extensive overnight experiments with different setpoints, the PID controller consistently maintained temperature within the desired range, confirming its effectiveness. Additionally, when the setpoint was changed during an ongoing experiment, the system showcased remarkable agility in adapting to the shift, ensuring the new temperature setpoint was maintained without any adverse impact on the cells.
APUS: An Affordable Solution for Biotechnology Research
One major goal of ours in designing APUS was to provide our hardware platform at a low cost. Many elements of our design were fabricated using 3-D printing technology, keeping overall production cost at a minimum. The bulk of our production costs stems from the materials used to motorize our fluidic system.
Plate Readers: These models typically have limited features and are used for simple absorbance or fluorescence measurements. Prices for basic plate readers can range from $2,000 to $10,000 or more, depending on the brand and specifications.
Mid-Range Plate Readers: These plate readers offer more advanced features, including the ability to perform various types of assays, kinetic measurements, and fluorescence intensity measurements. Prices typically fall in the range of $10,000 to $30,000.
High-End Plate Readers: High-end plate readers come with a wide range of features, including temperature control, multiple detection modes (absorbance, fluorescence, luminescence, and more), automation capabilities, and compatibility with a variety of assay formats. These instruments can cost $30,000 to $100,000 or more.
Multi-Mode Plate Readers: Some plate readers are designed to perform multiple types of assays, such as absorbance, fluorescence, luminescence, and time-resolved fluorescence. These are often at the higher end of the price range, ranging from $40,000 to $150,000 or more.
Microplate Readers: These specialized plate readers are designed for microplate assays and can cost anywhere from $5,000 to $30,000 or more, depending on their capabilities.
Automated Plug and Playable Microfluidic System (APUS): Our uniquely customizable hardware device is capable of capturing fluorescence measurements operated by a fully-automated processing stream and user friendly GUI. Our adaptable system is also compatible with a wide range of microscopes. We estimate that our system would cost anywhere between $1,000 - $1,200. This price point is significantly lower than even the basic level plate readers.
Sharing our hardware platform with the Synthetic Biology Community
In addition to sharing our newly developed hardware with the Synthetic Biology Community on the global stage at the iGEM Jamboree, we aim to share our project design on the world wide web to reach an even wider audience.
Access to our GitLab Repository below will assist anyone interested in replicating or expanding upon on our established APUS technology.