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Enabling Decentralized Nucleic Acid Detection for Improved Access and Timely Results
Decentralized nucleic acid detection empowers individuals, improves access to testing, and provides timely results, which can positively impact public health, disease management, and healthcare efficiency.
This approach offers several benefits to improve accessibility and sustainability since plastic can be washed and reused.
By combining the thermocycler, imaging station, fluorescence detection, data analysis, and sample preparation into a single handheld device, it becomes possible to perform nucleic acid testing in a portable manner. This allows for rapid and on-the-spot diagnostics, making it valuable for applications such as field testing, point-of-care settings, or resource-limited environments where access to centralized laboratories is limited.
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.
1 Compact and versatile thermocycler and imaging station for nucleic acid detection
The CAD view of the thermocycler assembly. It has an imaging station for visualizing fluorescence using something like a cell phone camera. When the Taqman probe binds to the amplified sequence the color change goes from red to green. Picture of what the thermocycler device looks like. Its made using 3D printer parts (ie., heaters, thermistor, electronics and Arduino based firmware).
2 Example of raw imaging data for TaqMan PCR using cell phone camera
Example of what the amplfied signal looks like when running a Taqman probe based PCR. When the probe binds to the amplified target sequence the signal shifts from red to green which can be quantified.
3 Affordable pipetting tools for decentralized nucleic acid testing
The system presented comprises essential components for nucleic acid testing. It includes a reaction master mix tube designed to hold the sample, a mineral oil tube used to maintain the reaction mix at the bottom of the PCR tube during thermocycling, a pipettor tool for precise liquid handling, a wide-bore pipette for aspirating and dispensing mineral oil, and a small volume pipette for aspirating and dispensing the reaction master mix. Additionally, the system features PCR tubes specifically designed to fit in the PCRmon thermocycler. To facilitate the process, the mineral oil is layered on top of the aspirated reaction mix.
4 Statistical data demonstrating PCR on a thermocycler made out of 3D printer extruder
Example of Taqman PCR using a thermocycler made using parts of a 3D printer extruder
5 Low-cost manual pipettor design
The pipettor is able to securely attach to different types of pipette tips. By squeezing the top part of the silicone tubing, it is possible to aspirate and dispense very small volumes. To consistently aspirate the same volume, a marking on the pipette tip can be drawn as a reference.
6 Demonstrating precise aspiration of PCR reaction mix using a low-cost manual pipettor design
In order to transfer the reaction from the source well into the PCR test tube, the process involves aspirating and dispensing the sample. This example demonstrates the ease and accuracy of aspirating microliter volumes of the reaction mix (in this case, the volume aspirated is 10µl). To aspirate, start by squeezing the tubing at the top of the pipette using your index finger and thumb, reducing the volume within the pipette. Then carefully position the pipette tip into the tube containing the reaction mix, gradually releasing the squeezing pressure to create suction and aspirate the desired volume until it reaches the marked level. Next, move the pipette tip to the empty PCR tube and dispense the aspirated volume.
7 Mineral oil liquid handling for PCR amplification process
Due to the absence of a heated lid in the PCRmon thermal block, the addition of mineral oil is necessary to ensure the reaction mix remains at the bottom of the PCR tube during thermocycling. As the mineral oil has a relatively viscous consistency, smaller pipette tips are not ideal for aspirating it, and instead, a larger bore pipette tip is more suitable. The precise volume of mineral oil added to the reaction mix is not critical. It is recommended to aspirate the total amount of volume, simply by squeezing and releasing the pipette entirely while the tip is submerged in the mineral oil. This typically results in approximately 30μl of mineral oil in the tube.