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Recently we have begun to see the emergence of high-volume, high-value applications of microfluidics for diagnostics. However, the commercial origins of microfluidics go back to the late 1970s. Why was so much promised so early, and why did it take so long to realise this promise? Often described as a disruptive technology, this aspect helped fuel the hype but at the same time it slowed commercialisation.

Economic stress in global health markets gives an increasing urgency to develop novel, cost effective solutions. Commercially viable immunochemistry, molecular and even cellular-based diagnostics are emerging to address real patient needs. More importantly, business models are emerging that support the commercialisation of these new devices.

As the market evolves those business models with optimal integrated design and manufacturing strategies will succeed, as well as those that seek to exploit the data generated by these lab-on-a-chip devices.

40-year Commerc ia l H i s tor yImperial Chemical Industries (ICI, 1926–2008) was one

of the largest manufacturers in Britain, with substantial business in chemicals, paints, polymers, pharmaceuticals and explosives. In the late 1970s, a group of researchers at its vast Winnington Research labs (now owned by Tata Chemicals) postulated that managing highly exothermic chemical reactions in micro-volumes would result in greater control of byproducts and began working in microfluidics. Today glass and stainless steel microfluidic reactors are used by industrial R&D chemists to understand the kinetics of unstable reactions (1).

In 1982 USA researcher Kurt Petersen published a paper suggesting that silicon, well established in the computer chip industry, could be used as a mechanical material (2). The paper suggested the design of a gas chromatograph etched into a silicon wafer. Although the device never worked, silicon technology became the workhorse for many microengineered devices, including ink-jet printer nozzles. Microfluidic devices are often called microfluidic chips, a carryover from the early influence of computer chip manufacturing technology. Semiconductor factories are often called Fabs, and this is the origin of our company’s name, MiniFAB.

Petersen went on to become a key entrepreneur in Silicon Valley. In 1996 he co-founded Cepheid, a company in the right place and at the right time when, post 9-11, it became urgent to detect anthrax spores in the US postal

system. From 1999–2002, Cepheid had been advertising on its website that it had a handheld DNA detection chip almost ready for commercialisation. The system that was hurriedly delivered to the USA postal authorities was as big as several domestic refrigerators; however, it was a success and formed the basis of strong business growth. In September 2016, Danaher Corporation announced its intention to acquire Cepheid for US$4 billion.

Academic Re search in M i c ro f l u id i c sIn the meantime, academic activity in microfluidics was

increasing. George Whitesides had suggested a relatively cost effective way for making microfluidic devices based upon casting using polydimethylsiloxane (PDMS) (3). Easily performed by graduate students, these elastomeric devices can be cast from any structure that has the inverse microfluidic features. With plasma activation of the surface, PDMS chips can be sealed to a glass microscope slide with adequate adhesion. The material is optically transparent and does not fluoresce under ultraviolet light, making it ideal for investigation of many bioassays.

Unfortunately PDMS has many shortcomings that make it unsuitable for most commercial applications. The material is not dimensionally stable and it strongly absorbs liquids. This provided a market opportunity for the commercial provision of rigid (thermoplastic) microfluidic chips or glass chips to academic researchers. Catalogues of common microfluidic structures are available from several companies. Although useful for lab-on-a-chip research, these standard configurations have limited use in developing integrated diagnostic devices, leading some to comment that they are a chip-in-a-lab rather than lab-on-a-chip.

Min i FAB a s M i c ro f l u id i c S e r v i c e Prov iderIn 2002, our academic research group at Swinburne

University of Technology partnered with local investors to create MiniFAB. We use our polymer microengineering understanding to translate clients’ assays into cost effective in-vitro diagnostic devices.

Fourteen years later, and having completed more than 1,000 projects for more than 250 clients around the world, MiniFAB has shown the viability of what is known as the ‘service business model’ for the contract development and manufacture of microfluidic diagnostic products.

Currently employing over 130 people, MiniFAB operates a cleanroom-based ISO13485 manufacturing facility that produces and exports more than 7 million nano-precise microfluidic diagnostic devices each year (Fig. 1).

Microf luidics Hype and the 40-year Commercial i sat ion Process to Diagnostic Devices .

Why so Long and What i s Next?Erol Harvey*

*MiniFAB, Scoresby, VIC 3179*Corresponding author: erolharvey@minifab.com.au

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What i s Spe c i a l About M i c ro f l u id i c s ?When dealing with fluid volumes that are in the nano to

low-milliliter range, the dominant forces that determine how the system behaves are those to do with surfaces and interfaces, and less to do with bulk effects. For example, mixing in channels is determined by diffusion rates since flow volumes are too small to create chaotic vortices relevant in shaking or stirring. Gravity is less important, capillary forces and surface contact angles can drive fluid flow. Air becomes one of the fluids in the system and can be manipulated to drive flow, separate volumes or sequence processes.

Surface-to-volume ratios increase considerably since surface scales as the square of dimension, and volume scales as the cube. Surface-dominated processes such as catalysis, capture and filtration become easier to make reproducible and it takes less time for the full volume to interact with the surface.

From an engineering perspective, the ability to reproducibly control the physical parameters associated with assays is a huge attraction, removing operator-dependent techniques such as shaking, stirring and pipetting. There is also the obvious advantage of reducing volumes of expensive reagents.

App l i ca t i on Fo cu s f o r M i c ro f l u id i c s Evo l ve sIn the early days of microfluidics, its application to

chemical synthesis, or blood chemistry analysis that drove much of the development. For microfluidic chip developers, key technical challenges at this time focussed attention on mixing strategies, valve design, microreactor design and cost reduction in fabrication.

As developers started to transfer lab-based assays into microfluidic chips, it was immunoassays, often using an adaptation of the ELISA format, that was most amenable.

More recently, molecular diagnostics has become the hot area for commercial development by MiniFAB. The ability to use amplification techniques and in-situ hybridisation provides a high-sensitivity and highly selective method for detecting target pathogens.

The current focus on circulating tumor cell capture and measurement for oncology applications has given a focus

for several new companies to raise capital and develop potential microfluidic solutions. MiniFAB develops and manufactures organ-on-a-chip devices, targeting the needs of drug discovery businesses wishing to improve screening techniques by addressing cell populations that behave more as organs rather than individual cells.

And in the research field we are even seeing complete organisms being grown in flow-through microfluidic chips. Publications have reported small organisms such as coral-on-a-chip (4) and zebrafish embryos in rather aptly named projects called fish-on-chips (5).

Paper-ba sed M i c ro f l u id i c sRecently many researchers (6,7,8) have become keen

advocates for paper-based microfluidics, and there is considerable academic research activity in this area. The wicking properties of paper are used to drive fluid flow, and simple wax printing can be used to define flow channels in the paper. Researchers argue that paper costs are much lower than polymer or glass, so these devices would therefore be cheaper.

This claim is yet to be proven, and as yet there are no commercially viable paper microfluidic diagnostic devices. Typically, the polymer represents less than 10% of the cost of a polymer diagnostic, with the majority of cost determined by reagents, the labour involved in its production, the amortisation cost of the production machinery and quality control. Although paper would reduce the cost of the base material, it is highly variable between batches and would need careful control of its properties. Therefore the increase in QC cost may offset the small saving in material costs.

Regu lator y I s sue s for D iagnost i c Deve lopmentThe first efforts at running ELISA assays in microfluidic

consumable devices produced poor results in comparison to lab-based instruments that form the benchmark against which point of care (PoC) devices are measured. Some thought that there would be a market for low-cost, field-deployable ‘indicators’ providing a traffic-light type output:• Definitely POSITIVE, • Definitely NEGATIVE, or • Go and do a better test.

In practice, the process of validating the new diagnostic in clinical trials almost always involves comparison to a lab-based benchmark. The regulators have resisted the idea of approving a device that is demonstrably poorer in performance than the current standard, even if an argument about rapid low-cost measurement could be made. Therefore, none of these products made it to market and it is now essential that any diagnostic device performs at least as well as, if not better than, the benchmark lab standard.

Another key regulatory hurdle from a commercial perspective is the attainment of a CLIA Waiver status from the US regulator – the Food and Drug Administration (FDA). CLIA is shorthand for the Clinical Laboratory Improvement Amendments. It requires that clinical laboratories be certified by their state as well as the Centre

Fig. 1 V iew of MiniFAB microfluidic production facility.

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The ALURE of MassifiedUndergraduate Research Spe c ia l Te chn i ca l Fea ture

for Medicare and Medicaid Services (CMS) before they can accept human samples for diagnostic testing.

For a PoC device to be truly for home use, it must be awarded CLIA Waiver status, and this is the target market for many of MiniFAB’s clients. This means that any untrained person can use the test and obtain a valid result, and that the risk of error is negligible. This is a significant technical hurdle for the engineering design of a diagnostic, as even the home pregnancy-test kit is often mishandled or used incorrectly.

Immunoas say D iagnost i c sThere are many enzyme-linked immunosorbent assays

(ELISA) (9) and these can be transferred to a microfluidic environment with little change. In a fully integrated microfluidic cartridge, assay sequencing, reagent mixing, incubation timing and washing can all be reproducibly controlled. In a good design, the user interacts with the device as little as possible – ideally only when inputting the sample and reading the result. This means that all reagents must be stored on the cartridge, either as dried-down reagents or as wet reagents stored in blisters or other chambers. Users do not want to be constrained to refrigerated storage, and validating 18-month shelf-life in ambient storage conditions is challenging.

Lateral flow systems are an alternate platform for these assays, and are usually lower in cost. However lateral flow systems are more difficult to multiplex and cannot support the sample cleanup and pre-processing possible with microfluidics.

Molecu lar D iagnost i c sThere has been a huge advance in the understanding

of molecular biochemistry, and many tools for handling, manipulating, creating, amplifying or modifying DNA and RNA are available. Understandably therefore, there is considerable commercial activity in translating this understanding into simple-to-use diagnostic devices with excellent sensitivity and selectivity, and much of MiniFAB’s current work is in this area.

The industrial community was keenly watching for a significant milestone – the first molecular diagnostic to be CLIA Waived by the FDA. This was achieved by Alere in January 2015 for their test for influenza A and B. The system was designed and developed by Axxin in Melbourne and is manufactured in Australia for Alere. In the race to compete, Roche purchased iQuum for $460M in April 2014 and a year later had the competing Cobas Liat system CLIA Waived for influenza A and B. Today there are six different molecular diagnostic systems that have been CLIA waived, and many more in the development phase.

PCR amplification requires thermal control of the system, and this requirement for an energy source, as well as timing and sequencing, usually means that molecular diagnostic microfluidic systems need to be designed to interact closely with a reusable instrument.

In an example of a fully integrated molecular diagnostic device, MiniFAB developed a cartridge that accepts

four independent sample swabs and performed lysis, DNA extraction, PCR amplification and Capillary Electrophoresis for separation and detection. The output was compared to the DNA forensic fingerprint library, and results could be obtained in as little as 40 minutes (10) (Fig. 2).

Data Management – Who Wants the In for mat ion and Who Wi l l Pay?

As a final output these devices simply produce data. People will only buy and use these units if they find the data useful and good value for money. An explosion of health data is an inevitable consequence of the rapid development of PoC diagnostic devices and the ‘consumerisation’ of health, whereby individuals purchase and use their own devices. How will this data be stored? Who will own it? How can value be extracted from the data?

These and many more questions related to so-called ‘Big Data’ are still wide open. What is self-evident is that if collected correctly, and linked to patient outcomes, this information will be a rich source for computer-based learning algorithms to analyse and generate improved diagnoses on the basis of prior data. This experience-based feedback loop is of course how medical practioners learn their art. In a world in which the microfluidic diagnostic is simply a peripheral, a hardware link between the patient and a cloud based diagnosis system, we could imagine huge advances in the accuracy and speed of response of diagnosis and treatment.

While a tantalising dream, this scenario is still a long way from reality. For a start, our regulatory system has no process by which to approve a device that improves its performance the more it is used. Approval is only given for diagnostics with a validated clinical response and a locked-down manufacturing process. For me, the next significant milestone to watch for in this industry will be when the first company manages to obtain regulatory approval for a diagnostic device that uses a cloud-based, self-learning algorithm for its output.

Fig. 2 Four-channel microfluidic device for DNA fingerprint analysis.

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Why i s i t so Hard?A diagnostic assay that works most of the time in the research lab is still

a long way off from being something upon which a life and death medical diagnosis can be made. If you have ever found yourself wondering if the reason why your lab assay hasn’t worked this time could “possibly be because the reagents are old – I’ll have to re-order” this is likely a sign that you do not fully understand your process. As physicists and development engineers, it is our job to find out what went wrong and fix it. As a company it is then our task to convince the regulator that we have a 100% reliable system. And having worked so hard to solve these problems we would probably not publish our knowledge in a patent for all to see. Instead we might hold that information as a trade secret, build a factory, employ some people and manufacture some products that we would sell to the world. At the end of the day, selling a robust, trusted diagnostic, one that is the very best we can possibly make, is truly satisfying and a huge motivation for all of us at MiniFAB.

…and Last ly, What About Theranos?The story of Theranos is still unfolding, and will likely make a good

Hollywood movie sometime in the future. Theranos promises cheap, rapid blood tests that, at least to those of us in the industry, sound too good to be true. With an engaging personality, a few good TED talks and influential high-net-worth friends, founder Elizabeth Holmes managed to raise $750 million to get her company started. Theranos is a private company and does not have to publically disclose financial information, nonetheless others valued the company at $9 billion at its peak, and more recently back to $750 million.

By the time this article is published we may know more. At the time of writing, US Federal prosecutors have launched a criminal investigation into whether Theranos misled investors about the state of its technology and operations. The quantum is large, but the story is not new. The promise of affordable, effective, pain-free health is a deeply human desire. How will the Theranos story impact future investment in diagnostics? Well, back in the Wild West, snake-oil salesmen tapped into the same human desire, but their fraudulent activities did not prevent the establishment of a global pharmaceutical industry. People will always be desperate to believe, but at the end of the day, a snake-oil salesman is still a snake-oil salesman and there seems little cure for that.

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1844. Shapiro, O.H., Kramarsky-Winter, E., Gavish, A.R., Stocker, R., and

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Saul, D.J., Chassagne, L., Landers, J.P., and de Mazancourt, P. (2014) Lab Chip 14, 4415-4425