Moving Toward Rapid And Low-Cost Point-Of-Care Molecular Diagnostics With A Repurposed 3D Printer And RPA

Traditionally, most nucleic acid amplification-based molecular diagnostic tests are done in centralized settings. In recent years, point-of-care (POC) tests have been commercialized for use in low-resource settings away from central laboratories. While most experts agree that POC molecular tests are greatly needed, their availability as cost-effective and easy-to-operate tests remains an unmet goal.

In a recent paper published in Analytical Biochemistry, a startup company in College Station, Texas presented a novel idea of converting a low-cost 3D printer into a device that can perform molecular diagnostics, including nucleic acid isolation (e.g., DNA/RNA) which is often the most difficult aspect of the diagnostics protocols, and thermal nucleic acid amplification using a technique called Recombinase Polymerase Amplification (RPA) developed by TwistDx, a UK company.

First, the reach team repurposed a 3D printer as a platform that can carry out medium-throughput, rapid, and high-performing nucleic acid extraction.  In 3D printing, users often operate printers via open-source host software such as Repetier-Host (Hot-World GmbH & Co., Willich, Germany) and Cura (Ultimaker B.V., Geldermalsenhe, the Netherlands) to slice the 3D model drawings and convert them into G-code, the language the 3D printer speaks. Next, the G-code is sent to the 3D printer via a laptop or an SD card.  To repurpose the 3D printer for nucleic acid extraction, the research team wrote G-codes to control the motion and many other functions of the 3D printer (e.g., controlling the temperature at the extruder and the heated bed) to perform extraction protocols with commercially available nucleic acid extraction reagents.

The modified printer shown in Fig. 1 of the paper can process up to 12 samples simultaneously in under 13 minutes for extraction (5-min lysis and nucleic acid binding, 3-min washing, and 5-min nucleic acid elution).  As typical commercial sample preparation equipment can cost up to $25,000, the 3D printers the team converted to carry out DNA/RNA isolation cost as little as $399.  Using converted 3D printers, high-quality nucleic acid extractions can be accomplished at a cost likely less expensive than the annual service contract needed for the high-cost extraction equipment.  After this paper is published, the research team purchased one of the lowest-priced ($160) preassembled 3D printer found on the market and has successfully converted it to perform DNA extraction.

With the ability to obtain purified DNA/RNA templates, the team demonstrated these templates can be rapidly amplified by isothermal nucleic acid amplification techniques using the 3D printer’s heated bed (Fig 2, Fig. 3 in the paper).  S. enterica (ATCC 19585) was spiked in 2% reduced fat milk at different concentrations (final concentrations of 0, 103, 105, and 107 CFU/mL), in which 100 μL was used in each extraction and the templates were eluted and detected by RPA.  The extracted templates were incubated together at 40°C in an aluminum block placed on the 3D printer’s heated bed, and their signal was monitored by a USB-powered detector.

Alternatively, the team used a $20 thermos container to carry out isothermal amplification (Fig 3, Fig 2 of the paper) after extraction with a repurposed 3D printer. Uncapped vacuum-insulated, stainless-steel thermos food jars can maintain water at elevated temperature (e.g., at 40°C needed for RPA) for over an hour which is long enough for multiple runs of RPA reaction.  A layer of clear mineral oil acts as a lid to minimize heat loss and allows for real-time reaction monitoring using a cell phone placed above the thermos to take pictures at 30s intervals.  Six consecutive runs of isothermal amplification and detection of Salmonella DNA by RPA was accomplished and presented using a thermos food jar.

The group is currently working under a Phase II NIH Small Business Innovation Research grant to develop an instrument for Chlamydia trachomatis and Neisseria gonorrhoeae diagnostics using the same 3D printer platform but with a cartridge designed for molecular diagnostics application.  This low-cost approach does not use complicated and high-cost components, making it suitable for resource-limited settings. When commercialized, it will offer simple sample-to-answer molecular diagnostics.

These findings are described in the article entitled Moving toward rapid and low-cost point-of-care molecular diagnostics with a repurposed 3D printer and RPA, recently published in the journal Analytical Biochemistry. This work was conducted by Kamfai Chan, Pui-Yan Wong, Chaitanya Parikh, and Season Wong from AI Biosciences, Inc.

About The Author

Season Sze-Shun Wong

Season Sze-Shun Wong is the co-founder of AI Biosciences. He has over 13 years of experience encompassing a broad range of disciplines including analytical chemistry, electrochemistry, biosensors and chemical/biological defense. He has directed multidisciplinary teams of scientists and engineers focusing on the development of integrated biodetection systems for clinical, environmental, forensic, and homeland defense applications. As a Principal Investigator, he has initiated and successfully secured SBIR and STTR awards that funded over $10 million of research during his professional career.

Speak Your Mind!

READ THIS NEXT

Can You Mix A German Shepherd With A Wolf?

Is it possible for wolves and dogs like german shepherds to interbreed and create a hybrid species? The short answer is yes, though to understand why it is beneficial to go into the evolutionary history of wolves and dogs. The History of Dogs And Wolves Wolves, like dogs, are members of the genus Canis and […]

Towards The Engineering Of Living Cells That Mimic Computers

Synthetic biology is challenging our current conception at multiple levels with an unprecedented potential. The aim is the (re)engineering of gene regulatory circuits to end with cells (re)programmed on purpose for biotech/biomed applications (viz., to implement novel or discover natural functions). This is accomplished by introducing suitable synthetic DNA pieces, mostly designed from scratch thanks […]

Exploring Boron Isotopes In Indian Thermal Springs

Geochemical analysis gives us a clear idea about the various chemical elements present in the rocks and soils through which the water flows and acquires certain elements from the rocks and in some cases precipitates certain elements. Geochemical interaction takes place at certain temperatures. If the temperature of a reaction is high, then more elements enter […]

Integer Aperture Estimation In The Presence Of Biases

Integer aperture estimation, also called integer ambiguity resolution, is a critical step to realized fast and precise global navigation satellite system (GNSS) positioning. It is a process which projects a float ambiguity to an integer ambiguity and then judges whether to fix that integer. Decades of research offer many projection methods. Efficiency and success rate […]

Climate Change Likely To Produce More Intense Rainfall And Landslides

Whether you believe it or not, climate change is unequivocal and it’s mostly our fault. According to Dr. John Cook and coauthors, who compiled a statistical analysis on the scientific consensus over an anthropogenic cause of climate change, over 97% of scientific papers expressing a position on climate change agree that mankind is to blame […]

Stimulation Of The Prefrontal Cortex Found To Reduce Violent Impulses

The prefrontal cortex is an area of the mammalian brain that covers part of the brain’s frontal lobe. The prefrontal cortex is linked with personality, planning, decision making and moderating social behavior, and in a new study, it was found that stimulating the prefrontal cortex with electrical currents reduces the desire to carry out violent […]

The Human PYHIN Proteins: Our Cellular Guardians Against DNA Viruses

In order to survive in an environment populated by hostile viruses ready to parasite them, mammalian cells have evolved a sophisticated variety of constitutively expressed receptors to detect extra- or intracellular pathogen-associated molecular signatures. Upon activation by binding to their targets, such receptors trigger a specific intracellular signaling which leads to a potent production and […]