How Digital Microfluidics Performs PCR within Minutes

Over the last year, testing for COVID-19 has become universally accepted as a way to help reduce spread of the virus. As a result, many different test methodologies have emerged. According to the CDC, the?“gold standard” for SARS-CoV-2 testing?is reverse transcription polymerase chain reaction (RT-PCR).

Standard RT-PCR begins by making complementary copies of DNA from target strands of RNA through a process called reverse transcription. Next, multiple copies of these DNA strands are produced through thermal cycling: DNA double strands are denatured at a high temperature (typically 95oC) to produce single strands, followed by the binding of primers to the target DNA and extension of those DNA strands at a lower temperature (typically 60-65oC).

This thermal cycling process is repeated, exponentially producing millions of copies of DNA, which are then detected via fluorescence.

Factors Enabling PCR Results within Minutes on DMF

Digital microfluidics (DMF) technology?miniaturizes and fully automates RT-PCR protocols using standard laboratory chemistries. Our DMF platform has demonstrated?PCR within 5 minutes?through optimization of the following factors:

Low Droplet Volume

Digital microfluidics electronically manipulates discrete microliter-sized droplets of reagents and samples between heat zones, maintained at ~95oC and ~60oC, which enables the heating and cooling process essential for PCR assays.

Shuttling tiny droplet mixtures using DMF mimics the original PCR method as invented by Dr. Kary Mullis (Nobel prize winner for invention of PCR), in which the reaction mixture was transported between two heat baths. Microliter-sized droplets have a low thermal mass allowing them to be heated and cooled with no lag time. Heating and cooling, aka thermal cycling, can be ultra-rapid when heaters and sensors are in immediate contact with the droplets.

Built-in Heaters and Sensors on Cartridge

Generally, with other PCR platforms, heating and cooling are performed convectively or conductively with a heat block adding additional thermal mass, which slows down the thermal cycling considerably in comparison to DMF.

Digital microfluidic protocols for PCR?embed tiny heaters and sensors within the disposable cartridge. These tiny heaters create heat zones localized to the microliter droplet, further reducing the thermal mass and together with embedded tiny temperature sensors precise and ultra-rapid thermal cycling can be performed.

Easy Sample Preparation and Microliter-sized Reagents

In conjunction with ultra-rapid PCR, sample preparation should also be quick. Typically, respiratory and other physiological specimens require sample preparation to lyse the pathogens to release the nucleic acids, which are?further captured onto a solid phase such as magnetic beads.

Digital microfluidics allows for automatic and rapid sample preparation methods and enables concentration of all the RNA to be captured on the magnetic beads, into a single droplet, and eluted off into another droplet to perform RT-PCR.

DMF requires only miniscule amounts of reagents and, therefore, mitigates the impact of supply scarcities on the number of tests that can be performed. This is particularly beneficial during the current COVID-19 pandemic as reagent supplies have been unpredictable.

Publication: PCR in 5 Minutes using DMF

Baebies scientists have published several peer-reviewed articles on PCR using DMF technology to detect various pathogens including viruses, bacteria, and fungi. . In this?2020 peer-reviewed publication in?Diagnostics, PCR for congenital cytomegalovirus (cCMV) infection was performed on a DMF platform and cartridge within 5 minutes.

RT-PCR Testing with Digital Microfluidics

To address the critical need for accurate testing with rapid results, Baebies developed an RT-PCR test for using a digital microfluidics cartridge.

Contact us to learn more or bring testing to your lab.

Updated 8/8/2023