CONTEXT

Prolight Diagnostics wanted to translate their microELISA magnetic-bead immunoassay into a point-of-care system while retaining its lab-quality performance.

SOLUTION

TTP designed, engineered and tested an, adaptable microfluidic cartridge, along with a benchtop instrument, featuring innovative blood-to-plasma generation and a novel protein purification and concentration system, all within an easy-to-use format.​

RESULT

Two assays have already been translated using the prototype MicroFlex cartridge, which has excellent correlation to established lab-based tests, and a ‘sample-to-answer’ workflow that is optimised for point-of-care settings.​

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Bridging the gap from lab assay to point-of-care test

Diagnostic tools based on protein–antibody binding reactions – known as immunodiagnostics – have been around for decades. Lateral flow devices, which use capillary action to drive fluid through antibody-impregnated membranes, are a cheap and reliable solution for point-of-care testing. But some biomarkers, such as those for neurodegenerative or cardiac conditions, need higher precision and sensitivity. In a lab setting, this is typically achievable using magnetic bead technology on a microtitre plate, but bridging the gap between distributed convenience and lab-quality performance is a persistent challenge.

So when Prolight Diagnostics approached TTP wanting to develop a platform capable of high-sensitivity microELISA immunoassays in a disposable, point-of-care (POC) format, we knew that we had a challenge on our hands that would require close collaboration between our engineering and biology teams. Fortunately, we were able to make rapid progress by using our ‘Flex Membrane’ platform as a starting point. Although this was originally conceived for DNA sequencing sample prep, it provided a flexible fluidic handling device that could be adapted for running Prolight’s immunoassay.

Building a prototype

Starting from the Flex Membrane format, we were able to develop and build a prototype microfluidic device for Prolight Diagnostics, the disposable ‘MicroFlex’ cartridge.

This was capable of running standard magnetic bead-based immunoassay chemistries, and performing all the core liquid handling functions of a central lab robotic system, including effective mixing, washing and magnetic bead processing. Improved microfluidic mixing in the cartridge enabled shorter incubation times compared to lab-bench methods, and by screening different antibody-conjugated paramagnetic bead options, we were able to define the magnetic system requirements.

Meanwhile, we conducted user studies to shape the instrument design. A key requirement that emerged from these studies was that we needed to eliminate the transfer of the blood sample from the collection tube to the device, a common barrier to the adoption of POC platforms. Direct sample loading capability therefore became a focus, and we developed and equipped the cartridge with a vacuum-tube loading system.

This allows the blood collection tube to be simply clicked into place on the cartridge, with the system then removing the sample, generating the plasma, and dosing it. In this way, we ensured that the MicroFlex system would integrate easily with existing clinical blood collection practices and delivering central lab quality results.

Innovating design improvements – generating new IP

Initially, the assay struggled with whole-blood input, resulting in significantly lower sensitivity compared to plasma. We were able to address this by adding to the cartridge a 10,000 rpm centrifuge able to generate large volumes of high-quality plasma. This allowed our biology team to optimise the assay for increased sensitivity, as well as making it easier to translate other lab-based assay chemistries, which are typically developed against plasma samples.

This innovative solution has now been granted European patent protection and since the development of the MicroFlex system, it has been become clear how many other use cases might benefit from integrating a centrifuge within a fluidic consumable. Whether that be needing to generate larger volumes of high-quality plasma, handling more challenging sample matrices or even extracting cell-free DNA or micro RNA from whole blood inputs. TTP is now actively exploring such commercial applications to further enhance the value Prolight may obtain from this technology TTP developed for them.

The relatively large volumes of high-quality plasma that were now available opened up another possibility – concentrating the sample before the immunoassay reaction to help improve the sensitivity. To do this, we exploited the ionic charges on specific target proteins to first purify and then concentrate them, with our biology and  engineering teams collaborating closely to ensure the necessary microfluidics could be integrated into the device. As a result, we were able to achieve a greater than four-fold increase in sensitivity compared to using the raw plasma directly.

Assessing product performance

So far in this project, we have built working prototypes of the benchtop instrument and have produced and tested many hundreds of cartridges using a pilot production line. This has allowed us to demonstrate the performance of the MicroFlex cartridge for two immunoassays:

• C-reactive protein: Used to test for chronic active inflammatory disorders and bacterial infections

• Procalcitonin: Used to test for bacterial infections.

In each case, clinical samples were split, with half run on the MicroFlex and half on a commercial central lab platform. The results show excellent correlation across the sensitivity range (see plots below).

‍Lab-quality results within a POC device

The final design for the MicroFlex system that we delivered to Prolight Diagnostics stands out because of its ease of use. The patient simply loads a blood sample tube into the cartridge, which is then placed into the benchtop instrument, to return a lab-quality result within 20 minutes.

This integration of a high-sensitivity chemiluminescence assay, magnetic bead handling, and a blood-to-plasma centrifuge – all in a microfluidic cartridge that fits within the palm of the hand– is an excellent example of how collaboration between engineers and biologists can translate an established lab-based assay into a POC platform without the typical costly and time-intensive process of reformatting the assay workflow. To bring this technology to market, TTP is now working within a commercialisation agreement to explore partnership opportunities and finalise the development with interested companies.