LabPilot

Overview

Lambert iGEM has experienced the firsthand difficulties of tedious pipetting leading to inaccurate results. Last year, we performed Rolling Circle Amplification (RCA) reactions, and this year we are conducting experiments with CRISPR-interference (CRISPRi) and Toeholds; all these reactions require precise pipetting in small volumes, taking time away from research, further hindering our organization, and reducing our overall efficiency and productivity as a team. Issues like these inspired us to develop LabPilot, a frugal automated liquid handler that allows researchers to focus time elsewhere rather than running lengthy reactions.

Pipetting is a fundamental laboratory technique used to transfer precise volumes of liquid from one container to another. Consistent pipetting is essential for achieving accurate results, maintaining data integrity, and facilitating advancements in synthetic biology research. However, manual pipetting, the primary method utilized in most labs, limits throughput (the amount of liquid passing through a pipette) and is easily susceptible to human error. To resolve these issues, automated liquid handlers are designed to streamline the process of pipetting liquids in laboratory settings. Liquid handlers can be programmed to perform a wide range of pipetting tasks with high precision, accuracy, and efficiency, enabling high throughput experiments (Armbruster, 2022).

Unfortunately, current automated liquid handlers are difficult to use and cost anywhere between 5,000 and 300,000 dollars (Retisoft, 2021). To provide underfunded labs access to liquid handlers, Lambert iGEM developed LabPilot, a frugal liquid handler made by integrating 3-D printable parts into an Ender-3. Currently LabPilot costs less than 200 dollars to produce and has reached a checkpoint where it can consistently aliquot static volumes of liquid (see Fig. 1). The only costs required to construct LabPilot are the Ender-3, priced at 180 dollars, and the 3D printed parts which are at a very minimal cost. Eventually, we intend on improving the device further to accurately pipette complex wetlab reactions. LabPilot enables research to be conducted in an efficient manner, filling a gap between expensive commercial liquid handlers and cheap DIY liquid handlers in educational labs.

Design

To handle the micropipette, LabPilot utilizes 3D-printed parts and repurposes the XYZ motion from an Ender 3 3D printer. LabPilot’s 2023 design has been updated to use an Ender 3 printer as the chassis, eliminating the need to fully design and manufacture a custom 3D-printed structure (see Lambert iGEM Wiki Hardware, 2023). With this new design, the attached micropipette is able to move with a significant increase precision. To improve the new LabPilot design from last year, we followed the engineering design cycle: design, build, test, learn. This process enabled us to efficiently refine different iterations of our device until eventually reaching a successful milestone. For example, last year we designed all of the necessary components and structural pieces for LabPilot on Autodesk Fusion, then 3D printed and constructed the build. When we reached the testing stage, we noticed reoccurring issues with our moving axes caused by the inevitable imperfections resulting from only using 3D printed parts. By using previous failures as a learning opportunity, LabPilot’s design took a completely different approach, finally bringing us success.

Figure 1. LabPilot Time Lapse

Structure

Lambert iGEM decided that building upon a commercial 3D printer for pipette movement would grant more precision than using an entirely self-made and assembled structure. The X, Y, and Z axes are controlled by a total of 3 stock Nema 17 motors, which respectively control the left/right movement and the forward/backward movement of the micropipette. The extra motor that controls filament extrusion was repurposed and paired with a gear rack and pinion, which creates linear movement to push against the micropipette’s button and pipette accurate amounts of liquid. Currently, LabPilot is designed for use with Eppendorf micropipettes to ensure proper fit. However, LabPilot could become adaptable to various brands of pipettes by accounting for their unique dimensions in CAD software. A 3D printed bedplate (see Fig. 2) with indentations to fasten components is attached to the Ender 3’s base. For the sake of our testing, we taped the bed plate onto the 3D printer. In the future, we plan to install magnets into our bed plate which will stick to the screwheads on the Ender. We also attached a stationary arm (see Fig. 3) to the bed plate to eject the pipette tips. The micropipette moves into the arm while sequentially traveling upwards. This way, the tip is pulled off the micropipette and discarded underneath.

Figure 2. Bed plate

Figure 3. Pipette tip ejector arm

Molds

Lambert iGEM created customizable molds that snap onto the 3D printed bed plate of LabPilot so that the user can attach their desired components, further attributing to its modular design. The molds have pegs that securely fit inside engraved indentations on the bed plate (see Fig. 4), which fixes the molds in place and enables the LabPilot app to calculate their position. LabPilot has a mold that fits every micropipette tip box, beaker, microcentrifuge tube rack, PCR tube rack, and well plate.

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Figure 4. The molds attach to the bed plate and allow for components to be precisely attached

Micropipette

The extruder was removed from Ender 3 to allow the new components to be mounted. A 3D printed plate modified from Kopyl et. al, holsters a micropipette and screws onto the spot where the extruder was formerly attached. We modified it to fit our desired dimensions, as well as our new pipette mechanism. Initially, we experimented with industry-grade linear actuators in an effort to create an axis that pushes down on the pipette plunger. This posed many issues for us mainly because the Ender 3’s stock microcontroller is unfitted to send non-stepper motor pulses. From observing this, we understood that we would have to use a stepper motor for linear actuation instead. The most economical solution would be to repurpose the Ender 3’s extrusion stepper motor into a pipetting mechanism motor. This led to our approach at a gear rack and pinion design (see Fig. 5) to convert rotational force coming from the Nema 17 motor, which was formerly used for extrusion, into linear motion. The linear motion is used to push down on the micropipette’s button with precision, and can also be controlled the same way as the rest of the motors.

Figure 5. CAD model of the gear rack and pinion attached to the pipette holder (Left). Actual 3D printed design attached to the Ender 3 (Right).

LabPilot Construction Guide

We created an instruction manual that goes through the steps needed to construct LabPilot from scratch. All CAD models can be downloaded from our GitLab here.

App

In a continuation from last year’s project, Lambert iGEM is designing a web app to control the liquid handler in an effort to streamline the user experience for researchers (see Lambert iGEM Wiki Hardware App Section, 2023). Unlike the complex software that commercial liquid handlers such as Opentron operate on, the LabPilot app has a simple, human-centric user interface designed to be used immediately with simple and intuitive commands. This design enables users to quickly start using LabPilot and efficiently simplify their tasks without any difficulties.

How it Works

To control its actions, connect the Ender 3’s stock controller board to a laptop running the LabPilot web app via USB. The microcontroller moves the micropipette to a specific location by using a virtual coordinate system, which defines different areas of the surface with numbers. The LabPilot app knows what actions to take in order to move the desired axes to their coordinate destination. By utilizing a system where the app executes high-level operations by compiling pipette actions into low-level instructions for the microcontroller (see Fig. 6), users have complete control over their LabPilot device.

Figure 6. LabPilot’s web app turns simple commands into low-level G-code instructions.

How to use

To use LabPilot, users must connect a laptop to LabPilot via a USB cable and open its web dashboard. Once the dashboard is open, there is a simple setup process where users can drag and drop components from the sidebar to recreate the physical layout of their LabPilot device (see Fig. 7). After setup, the app digitally recreates the layout of this device from data gathered in the setup process, enabling adequate simulation and control. Then, the users can queue and perform pipetting actions.

Figure 7. LabPilot’s setup interface.

All pipetting actions consist of a reagent source and a dispensing source. Users can click on any well or beaker to select a reagent source. After choosing a reagent source, users can select a dispensing source by clicking on any well/beaker or multiple dispensing sources (by clicking and dragging or holding “shift” while they select multiple sources). Afterward, users will select the amount they want to dispense to each dispensing source and can add it to the queue. All pipetting actions in the queue are performed in the order they were assigned but can be dragged around to reorder in the queue.

Results

To validate both the consistency and accuracy of LabPilot, Lambert iGEM had LabPilot aliquot 15 microliters of water into 4 tubes, combining all axes’ movement as well as the tip attachment and ejection. The aliquot, using a new tip for each tube, was controlled by a temporary program that turns written G-code into axis movement. Eventually, the web app will be able to generate the G-code itself to perform these procedures. The data was measured by weighing the tubes before and after the trials on a Mettler Toledo analytical balance with +/- 0.1mg accuracy (see Table 1). The experimental results show an average error of 9.32%, or 1.38ul. The comparison clearly shows LabPilot’s consistent functionality which is sufficient for aliquoting. The results of LabPilot’s pipetting demonstrate LabPilot’s readiness to be implemented in a laboratory setting.

Conclusion

Researchers and educators can leverage LabPilot’s capabilities to automate tedious pipetting tasks, enabling them to focus on higher-level analysis and interpretation of results, accelerating the pace of scientific discoveries. LabPilot upholds the necessary standards to be viable for allotting liquids as seen through Lambert iGEM’s proof of concept. In the near future, Lambert iGEM plans to develop a kit available for purchase and make LabPilot commercially available. Furthermore, LabPilot’s next checkpoint will be achieved when it can reliably conduct reactions far more complex than simple single-volume procedures. Some examples of complex reactions LabPilot could eventually execute would be DNA sequencing, sample preparation, high-throughput screening, and assay development. With sufficient aliquoting capabilities, LabPilot’s cost-effectiveness (at only $200), user-friendly interface, and open-source nature make it accessible and adaptable to all laboratory settings.

References