Marc Green reveals how POC diagnostics is benefiting from electrochemical PCR technology.
It is now well established that on-demand, actionable test results delivered by point-of-care (POC) diagnostics can better inform clinical decisions and improve patient outcomes. Although to date, the availability of high-quality POC tests for many infectious diseases has been limited. State-of-the-art high-sensitivity laboratory tests utilise nucleic acid technologies, which have proven difficult to adapt to a format suitable for use at POC.
However, electrochemical polymerase chain reaction (PCR) is an innovative technology that enables rapid, easy-to-use nucleic acid-based testing for infectious diseases at POC.
The majority of nucleic acid tests today use optical detection, relying on expensive, fragile optical sensors that require careful routine calibration. This presents a problem in POC settings where equipment is subjected to frequent relocation, while routine calibration imposes additional duties on already busy clinical staff.
Diabetes monitoring Electrochemistry has no such constraints, requiring only a low cost potentiometer, best demonstrated by the successful use of the technology in blood glucose meters for diabetes monitoring.
Electrochemical diagnostic devices are extremely robust and require only a simple self-check to ensure that the system is functioning correctly. This is check is performed under complete software control, removing the need for any user calibration.
Any diagnostic device for use at POC should perform all aspects of the test process rapidly and with minimal user interaction.
For example, the Atlas Genetics system requires only raw clinical specimen (such as urine, swab extract, etc.) to be added directly in to a port on the test cartridge, the cartridge is then loaded on to the instrument where the test type is determined and the test is performed entirely within the cartridge, under complete instrument control (Fig. 1).
DNA in the sample is extracted and purified and then a section of the target DNA is amplified using a highly optimised, ultra-rapid PCR reaction.
The speed of the DNA amplification is achieved using an extremely fast Peltier heater and thin-film laminate construction of the PCR chambers, allowing rapid heat transfer.
The amplified product is detected electrochemically using an electrochemically-labelled DNA probe that hybridises to the amplified target DNA. The resulting double-stranded DNA complex is digested by a double-strand specific exonuclease enzyme, which releases the electrochemical label.
This occurs in situ with a screen-printed carbon electrode. A voltage applied to the electrode oxidises free electrochemical label releasing electrons and generating a current that is measured by the system potentiometer (Fig. 2).
In negative samples, where there is no target DNA, the probe remains single-stranded and is not digested by the double-strand specific exonuclease, therefore no electrochemical label is released and no current detected.
Electrochemical labels (which are typically based on ferrocene) can be synthesised to oxidise at a specific voltage, something that’s referred to as the oxidation potential (Eox). In the Atlas Genetics technology, electrochemical labels covering a wide range of Eox are produced by adding different functional groups to the core ferrocene structure.
Multiplexing, to enable a number of disease targets to be detected simultaneously in a single test, is achieved using specific probes labelled with different electrochemical species.
The current generated at the Eox of the probe is then measured to determine the presence or absence of a DNA target (Fig. 3).
For more information visit www.scientistlive.com/eurolab
Marc Green is with Atlas Genetics in Wiltshire, UK.