RPA and microfluidics
Antibiotic resistance is a major, growing threat to human health worldwide. There is an urgent need to develop easy-to-use, sensitive tests that can detect drug resistance at the bedside so that appropriate, potentially life-saving antibiotics can be prescribed without delay.
However, traditional clinical tests used to determine antibiotic resistance are based on cell culture techniques or on polymerase chain reaction (PCR) assays, neither of which is suitable for point-of-care diagnosis.
A team at the University of Southampton led by professor Hywel Morgan has now developed a cost-effective, highly sensitive and portable digital microfluidic device that exploits Recombinase Polymerase Amplification and on-chip fluorescence to quantitatively detect antimicrobial resistance genes in DNA extracted from patient samples. The researchers hope that the platform could form the basis of an easy-to-use, disposable device that hospitals or community doctors could use routinely to identify multiple antibiotic resistance genes in patients with serious infections, so that the optimum treatment can be started as soon as possible.
The digital microfluidic platform exploits thin-film transistor (TFT)-controlled electrowetting-on-dielectric (EWOD) technology, or active matrix EWOD (AM-EWOD). The AM-EWOD chip developed by the Southampton team comprises 16,800 individually software-controlled electrodes that can manipulate nanolitre-scale droplets containing reactants, sample, and controls, in two dimensions, as well as define droplet size and shape. The chip also includes a built-in impedance sensor for real-time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater to keep the droplet temperature at the optimum 39°C temperature for the RPA and microfluidic reaction.
The researchers used their prototype AM-EWOD device to amplify and detect the extended spectrum β-lactamase resistance gene blaCTX-M-15 in DNA extracted from E. coli bacteria. To use the chip, droplets of DNA, RPA reaction mix, magnesium acetate and control are first loaded by pipette into reservoir electrodes, and then aliquots of these, or daughter droplets, are transferred from the reservoirs under software control to predetermined positions on the chip and mixed using a programmed mixing sequence so that the RPA reaction can proceed. After amplification another software controlled sequence transfers the droplets to the fluorescence detection position on the chip.
The reaction droplets are continually shuttled backwards and forwards to speed mixing and the RPA reaction. This continuous mixing improves target DNA detection 100-fold compared with a benchtop RPA assay, professor Morgan says. “The chip was capable of detecting a single blaCTX-M-15 gene copy within about 15 minutes.” Using nanolitre-volume droplets also means that about 50 times less reagents are used than are required for a conventional benchtop assay.
Professor Morgan’s team describes the technology in Lab on a Chip, in a paper titled, ‘Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform’1a. “Since publication of the paper we have carried out multiplex assays on the same chip, to detect three different bacterial resistance genes,” professor Morgan notes. “Feasibly a single chip could be used to both identify the organism that is causing an infection, and to determine whether that organism is resistant to certain antibiotics.”
In subsequent work the team has developed a chip device that combines a semiconductor TFT nanoribbon sensor with RPA to offer a pH-based electrical readout system. They claim that this approach provides even faster, more sensitive detection than conventional fluorescence using exo-probes for quantifying the amplified target DNA2. In tests the pH sensor technology was capable of detecting fewer than 10 copies of antibiotic resistance genes in genomic DNA extracted from E. coli or Klebsiella pneumoniae clinical isolates, within 15 minutes, and for 100 copies a positive result was achieved in under three minutes. Professor Morgan says the transistors are easy and inexpensive to produce, and could easily be incorporated into a digital microfluidic system. They report on the pH sensor technology in Biosensors and Bioelectronics, in a paper titled ‘Ultra-fast electronic detection of antimicrobial resistance genes using isothermal amplification and Thin Film Transistor sensors.’
1Lab on a Chip 2015. Jul 21;15(14):3065-75. doi: 10.1039/c5lc00462d
2 Biosensors and Bioelectronics 2017. Volume 96, 15 October 2017, Pages 281–287. doi: 10.1016/j.bios.2017.05.016
a This report is independent research funded by the National Institute for Health Research (Invention for Innovation (i4i) Programme grant II-ES-0511-21002). The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, National Institute for Health Research or Department of Health.