The longest microfluidic device in the world?

A new generation of miniaturised microfluidic devices is set to make multiplex quantitation of biomarkers affordable and portable

Currently, multiple biomarkers are measured routinely by healthcare providers for diagnosis of diseases, in particular cancer and cardiovascular diseases. However, until now the design of miniature portable devices for the detection of multiple biomarkers has relied on conventional microfluidic technology that presents major problems relating to the cost of manufacturing and scalability. Capillary Film Technology (CFT), a UK-based SME, has developed an approach for the production of multiplex diagnostic strips from a continuous plastic ribbon. The process allows the manufacture of plastic film that contains 10 microfluidic capillaries embedded along its length but can be produced in lengths of 10 kilometres or longer.

While the number of diagnostic tests is growing exponentially, healthcare budgets have become ever tighter. It is clear that testing at the point of care (PoC) offers both health and economic benefits, meaning clinicians save multiple consultations by performing a single rapid test in the surgery that results in an instant diagnosis. Furthermore, in emergency situations, such as suspected heart attack, testing rapidly would allow accurate and fast treatment, resulting in improved health outcomes as well as reducing the costs to the health system.

Over the past few decades, microfluidic technology has performed many impressive feats, in particular the miniaturisation of assays from test tubes or microwell plates to channels in the range of 50-500 microns. Diffusion times are reduced meaning that assays can be performed extremely rapidly. Typically an assay traditionally taking 3-6 hours in a microwell plate can be performed in less than 15 minutes in micro capillary film. While this is a nice concept, simple quantitative disposable devices containing microfluidic technologies continue to be slow to market as the associated analysis and fluid control of the microfluidic component still requires complex laboratory scale equipment such as pumps and microscopes.

At the other extreme, by far the most widely used PoC test format is the dipstick, where a test strip is dipped into the sample, or a sample is applied to a well at one end of a test strip. The test strips are typically a nitrocellulose membrane with all required antibody reagents deposited in the strip, and coloured lines indicate results. The best known example is the pregnancy test stick, but in recent years many new diagnostic applications have been applied to this ‘lateral flow’ technology. The scalable manufacture and very high volume of tests allows economical production of test devices. Consequently, lateral flow remains the mainstay of PoC testing, with the performance of lateral flow being pushed to its limit.

However two main weaknesses remain. Firstly, while qualitative “yes or no” results are rapidly delivered, a true quantitative measurement is challenging for lateral flow. More significantly, sensitivity is very limited as well as it being difficult to detect more than one target analyte. As the number of clinical biomarkers grows, there is an urgent need for devices with fully quantitative and multiplexing capability. In other words, they are unable to measure multiple biomarker analytes in a single sample whereas microfluidic devices containing micro capillary film will be able to deliver sensitive, quantitative, rapid multiplex testing. The key question is whether they can be produced at scale at an affordable cost that is competitive with lateral flow technology.

Invented by Professor Malcolm Mackley and Dr Bart Hallmark at the University of Cambridge in 2005, the micro capillary film (MCF) is a simple material comprising a flat ribbon of plastic typically up to 10mm wide and between 0.5-2mm thick that is produced by a novel melt-extrusion process. Melt-extrusion is perhaps the most cost-efficient method for embedding micro features within a thermoplastic matrix. While this sounds simple, this required a deep understanding of the rheology of polymer melts and expertise in extrusion manufacturing.

Many applications for MCF have been explored, including solar energy, Formula 1 racing, drug delivery, antibody purification, and flow chemistry. While these applications all exploit the material’s unique properties, they all share one key advantage: continuous manufacturing that offers the promise of affordable products in commercial quantities.

A key milestone in the development of MCF was made by co-inventors and founding directors of CFT, Drs Alexander Edwards and Nuno Reis while at the University of Cambridge. They discovered that, if made from the right plastic, MCF is transparent. While individual capillaries have been used to perform simple low volume assays for many decades, any colour change could not be measured at any level of sensitivity.

“If you have ever peered through a glass stirring rod you will be familiar with the problem. In essence, both the capillary tubing and the water filled channel behave like a powerful cylindrical lens when viewed from the side distorting any signals from them,” explained Dr Edwards. “To eliminate this problem, a very special family of plastics are used – fluoropolymers, that surprisingly have a refractive index identical to water. Fluoropolymer MCF, named FluorEx, is completely transparent when filled with water or aqueous assays such as immunoassays. This allows simple quantitative assay measurements to be performed with low cost digital imaging, for example using CCD or CMOS sensors, consumer flatbed scanner, or smartphone cameras.”

CFT is developing MCF using fluoropolymers for use in the diagnostic industry.

“All the established benefits of microfluidics are easily achieved while simply using existing conventional reagent chemistry,” said Dr Reis.

For example, a quantitative assay for the biomarker Prostate Specific Antigen (PSA) can be completed within 15 minutes, while the low background and unique optical transparency allows the detection to be extremely sensitive such that human inflammatory markers can be measured in the low pg/ml range using existing colorimetric assay technologies. Furthermore, as the MCF contains ten capillaries, this allows multiple measurements from a single sample whether by replicates or by adding different materials or capture antibodies into individual capillaries. Critically, this allows multiple analytes to be detected in a single sample termed 'multiplexing’.  This is all performed in a piece of film that is 0.5 x 3.0cm in size, similar in size to two back to back matchsticks. Consequently blood sample volumes are very low, less than 2ul to fill as single capillary.

CFT are currently building partnerships with diagnostic companies who have already identified critical applications where high performance scalable technologies are needed. In many cases, established diagnostic companies already have PoC devices and fluidic technology that can be combined with FluorEx to give a major boost in capability and performance. For example, multiplexing can be achieved in a previously single analyte system, or an improved quantitative readout can be delivered with analytes detected down to very low levels. Many digital readers have been produced for lateral flow tests which can easily be adapted for FluorEx based tests, allowing microfluidic performance to be delivered in a remarkably similar test format to LFA.

CFT are also working with NHS England. Around 100,000 people in the UK suffer from heart attacks each year while many more suffer chest pain and other symptoms of acute cardiovascular disease. CFT are investigating a solution to this pressing problem. Facilitated by the latest SBRI Healthcare competition, the NHS requires the development of a new diagnostic product that will improve UK health outcomes but in an economical manner. CFT are completing a feasibility study commissioned and funded by NHS England to evaluate whether accurate diagnosis of different types of acute heart problems can be achieved. Currently, a number of different blood tests are offered, each of which can diagnose different subtypes that are performed in central testing laboratories. By performing all tests rapidly from a single sample, a much faster diagnosis may be possible in an emergency setting. While in a GP’s surgery, those individuals who suffer the terrifying symptoms of a heart attack but may not have a serious heart problem, these tests should offer a rapid reassurance allowing them to return home safe in the knowledge that they don’t have acute cardiovascular disease. If CFT is successful with this feasibility study, this may lead to a full scale development programme that can deliver a new generation of cost-effective acute cardiac syndrome blood tests for the UK and worldwide.

The low manufacturing cost of MCF opens the door to developing affordable portable diagnostic technologies for low resource healthcare environments.

CFT is currently continuing to build diagnostic collaborations internationally to develop new applications and devices that will realise the potential of the micro capillary film. However, things won’t stop there. There are many other sectors to which the technology can be applied such as food testing, R&D and chemical industry.