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LabBot: Transforming 3D Printers into PCR Machines for Laboratory Automation

The system consists of a milled thermoblock and case for secure PCR tube holding, an illuminated tube holder for fluorescence imaging, an Arduino microcontroller shield for temperature control, a command-line Python-based software for thermocycling and imaging, reusable PCR tubes, mineral oil for heat transfer, a power supply for operation, and a pipettor module with pipettes for precise reagent dispensing.

Image 1 3-D printer robotic scheduler software TaqMan probe PCR layout
1 Robotic scheduler software for TaqMan probe PCR layout

The layout of LabBot deck that illustrates the objects used in the example Taqman probe PCR based nucleic acid detection assay. The LabBot Robotic Schedular software includes this tool where you can define objects to document your method.

Image 2 Design macro for mineral oil aspiration
2 Macro for mineral oil aspiration

Setting aspiration of mineral oil macro using the LabBot Robotic Scheduling Software

3 Step 1 Mineral oil aspiration

The LabBot Robotic Scheduler software makes it easy to assemble macros and save them so that you can point, click and run. Here is how an oil aspiration macro was saved, selected, and run.

Image 4 design pcr mastermix aspiration macro
4 Master mix aspiration macro

How a macro for sample aspiration can be designed for controlling a 3D printer

5 Step 2 Master mix aspiration

After the pipettes load the mineral oil, then the master mix is aspirated. They have to be done with these cut down PCR tubes in order to aspirate 10μl of master mix since these pipettes have a gasket membrane that is used for sealing tubes which are done afterward. Aspiration of 8μl of master mix. The tubes contain 10μl.

Image 6 LabBot robotic scheduler software - select and run tube loading macro
6 Step 3 Load tubes using pipette tips

After master mix aspiration the pipette tips are moved to PCR tubes so that they can be loaded and sealed for thermocycling.

7 Loading tubes so that they can be moved

Image 8 LabBot robotic scheduler software - dispense into tubes macro
8 Step 4 Dispense master mix and mineral oil into tubes

After tube loading, now the pipettes can dispense the master mix and mineral oil that it previously aspirated. Dispense master mix and mineral oil into tubes for thermocycling PCR detection.

9 Dispensing reaction mix into loaded tubes

10 Dispensing reaction mix into loaded tubes

Image 11 LabBot robotic scheduler software - thermocycling macro
11 Step 5 Thermocycle and imaging reaction

Now the samples and be incubated and it is possible to image after every cycle or every so many cycles. The macro does the imaging after 5 cycles (95C denaturation for 30 seconds and 65C extension for 2 minutes).

12 Thermocycling by moving the tubes around

Image 13 LabBot robotic scheduler - tubes removal macro
13 Step 6 Remove the tubes

After the reaction is finished, the system includes a technique for removing the tubes. Then the pipettes can be washed and on to the next set of samples. Tube removal technique. After the reaction is finished the tubes are removed using a removal tool

14 Removal of tubes after the reaction

Image 15 LabBot TaqMan Real-Time PCR example data
15 Example data from a Taqman PCR run on a modified 3d printer

When visualizing the fluorescence (using a blue LED and amber color filter in front of a camera). The Taqman probe oligonucleotide DNA sequence changes color from red to green when it binds to the target sequence. So the approach is to quantify the change of color of the course of the thermocycling process. Comparison between a template and no template is plotted to demonstrate how the PCR amplified nucleic acid signal can be detected using a Taqman probe.