Periodic Reporting for period 3 - DECISION (A minituarized disposable molecular diagnostics platform for combatting coronavirus infections)
Reporting period: 2022-07-01 to 2023-06-30
The aim of DECISION is to develop an easy-to-use, low-cost, disposable test for COVID-19 that can give reliable results, on the spot, within minutes. The idea is that it can be deployed anywhere; at drive-through testing centres, airports, physician offices, hospitals, quarantine centres, and eventually, even in people’s own homes.The overall deliverables of the project include the system integration, combining fluidic elements with electronics, system testing and verification, including the performance of the PCA®, fluidic functionality and reproducibility and compatibility with and robustness with real samples.Since it first appeared at the end of 2019, the SARS-CoV-2 virus has proven adept at spreading rapidly throughout entire populations causing Covid-19. To stop the virus in its tracks, we need to be able to quickly identify people who are infected, including those with little or no symptoms. Currently, the gold standard and the most reliable method of testing for COVID-19 is through a polymerase chain reaction (PCR) test. However, the test alone can take several hours to run, moreover, the samples must be sent to a laboratory, meaning most patients have to wait for at least a day to get their result. In that time, they either need to be in quarantine or possibly infect other persons if they are actually carrying the virus. Faster tests such as antigen tests do exist but are expensive and/or not always reliable. The diagnostic platform developed in the DECISION project will enable the rapid detection of COVID-19 infections on-site in any possible setting, including drive-through testing centres, airports, or medical offices, up to be used as a home test. The low-cost, miniature, disposable diagnostic platform is powered by a next-generation nucleic acid amplification technology called pulse-controlled amplification (PCA®), providing sample-to-answer workflows in less than 15 minutes.Like a PCR test, PCA® amplifies the genetic material of the virus. However, PCA® is both faster and cheaper, and the device itself can be build small enough to become portable. PCA® is a two-step PCR technique (the device cycles between base temperature and denaturation temperature) relying on pulsed electrical heating. With this approach cycle time is reduced to several seconds. Ultimately, the project aims that user will have to wait only 15 minutes to get their result.
The first-generation device developed since the beginning of the project is an essential basic microfluidic PCA®-chip with a reaction chamber containing the for the PCA needed special heating elements and basic microfluidic. The device can be connected to an i.e. laptop via USB2.1 with no additional electrical connections required. The amplification reaction is realised by passing electrical pulses through the heating element in a reaction chamber of a PCA®-chip, by generating local heat regions with very high temperatures for very short periods used for the denaturation steps needed in a PCR. The first-generation device contains electronics for optical dual colour real time detection. The built-in bicolor-LED-based detection unit was designed with excitation and detection channels situated at an angle to each other in the reflection mode. The excitation light is filtered with an optical filter to ensure that the wavelengths required for excitation of the fluorescent markers is emitted only. The fluorescence signals are processed by the specially written software and displayed as curves in real-time. The workflow for the lysis and RNA extraction was partly based on the existing protocol for Octea Device, which is a multiple sample desktop PCA®-device developed at GNA Biosolutions, wherein in particular a washing process including the transportation of RNA released from the virus from the incubation buffer to the PCA-mastermix was performed in the microfluidic chip. The microfluidic chip for this purpose was developed by Microliquid and consists of an inlet, microfluidic channels, a PCA®-chamber and an outlet. Additionally, there is a luer port fixed onto the outlet and magnetic stage where the chip can be placed so that magnetic beads which are used as target capturing and transportation means concentrate the target in the reaction chamber near the heating elements and ensure an efficient washing process with very little target loss. It was possible to show, that extracting genetic material from the sample containing down to 50 SARS-CoV-2 VP (virus particles). Investigations aiming to simplify the workflow were performed by IMB BW (Bundeswehr Institute of Microbiology) and GNA Biosolutions. A total number of 17 buffers was tested. We were able to identify three very promising candidates for simplifying the workflow and not inhibiting the PCA process. It was possible to omit a temperature step and reduce a significant amount of time. However, we have also seen that using human samples may still be challenging as they may contain substances in the samples influencing the performance of the modified buffers. The PCA® in the device was investigated using a well-shaped chip rather than the microfluidic chip described above in order to simplify experiments and due to lack of quantities. It was possible to show the amplification of SARS-CoV-2 target in concentration of 100VP in the sample. This concentration is below the average commonly known value for the SARS-CoV-2 viral load. A second generation device has already been developed. Although this device now has a slightly larger footprint, it offers much more options to control the experimental parameters, settings as well as readout. Multiple sensors on board enable the collection of a large amount of information during a PCA-run, which will help to understand and refine the process even more precisely.
The new disposable molecular platform will be a small electronic device with a PCA® chip and an easy to use sample preparation kit for performing the PCA®. The sample preparation and operating the disposable device will be self explanatory and easy enough to be performed by any healthcare professional. The individual steps of use will include taking the sample from mouth or nose using a swab, sample on swab is brought into solution using the simple sample preparation kit, sample solution is introduced into the reaction chamber of the disposable PCA® chip, PCA® is run, amplifying the target nucleic acid, optical detection and displaying result in less that 15 minutes PCA®. The disposable molecular platform has superior properties which will be a game changer for the detection of infected persons and containment of transmission, for Covid-19 and future epidemics. Due to the size and robustness, the low cost device may be used in various scenarios where the disposable device will bring the needed solutions. Possible scenarios include, in particular, doctors' offices, test stations, clinics and even home tests.