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Breaking new boundaries with microfluidics Kevin Land1*, Mesuli Mbanjwa1, Suzanne Hugo1, Jerry Chen1 and Klariska Govindasamy

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CSIR Materials Science and Manufacturing, PO Box 395, Pretoria 0001

*Corresponding author: Kevin Land: kland@csir.co.za

Reference: IN28-PA-F

Abstract Microfluidics is an important emerging research platform in South Africa. It deals with the control and manipulation of very small quantities of fluids (typically microlitre and smaller) inside micro-channels. Microfluidic-based devices show great promise for easy-to-use, inexpensive, point-of-care health diagnostics. Microfluidic devices also serve as excellent tools for the facilitation of micro reactions in droplets. The CSIR has taken the lead in establishing research and development facilities for microfluidics in these fields. We report on the facilities which are available and give an overview of the projects which are being undertaken in this field. These projects include the generation of emulsions for advanced particle production, the research into components to be integrated into an HIV/Aids diagnostic device and the development of a hand-held diagnostic device to be utilised for environmental and human health applications and other health-related issues. In addition, work on the development of conducting polymers, aimed specifically at reducing the number of components in micro fuel cells, is described. Finally, we report on the establishment of a Microfluidics Research Network in South Africa, aimed at creating the space for researchers involved in microfluidics and related fields to interact with one another, including the sharing of information, organization of specialised conferences and collaborations between researchers. 1. General introduction Microfluidics, a distinct new field in South Africa, focuses on the manipulation of fluids inside channels on the microscopic scale. It has the capability to significantly impact areas of biological analysis and chemical synthesis. The manipulation of fluids, particularly on a small scale and at very small volumes, offers numerous possibilities. The rapid growth in research and development of microfluidic technologies owes much to the numerous potential advantages offered should these technologies become mainstream. These advantages include:

• efficient use of expensive chemical reagents; • low production costs per device (devices can easily be mass-manufactured using existing

technologies); • scalable and flexible devices (as an example, chemical production can be scaled by stacking

individual devices); • faster•

analyses and results, including the possibility of real-time results; better control of process parameters during chemical reactions (droplets serve as excellent containers for precise control of mixing times and volumes); and

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• the elimination of the need for sample preparation (Sia and Whitesides 2003; Erickson and Li 2004).

In addition to the technical benefits described above, the technology is also attractive from a human capital development and scientific research perspective. It is a new and exciting technology, and due to its multidisciplinary nature, offers the opportunity for many researchers to be involved across a very broad range of disciplines. This is hugely advantageous to the CSIR and South Africa in general, as it offers the chance for universities, research institutes and industry to work together in developing cutting-edge technologies, which in turn will have direct a social and economic impact on the lives of ordinary South Africans. In this regard, the CSIR has taken the lead in establishing research and development facilities. This paper will provide an overview of the available research facilities and the current projects being undertaken in this field at the Materials Science and Manufacturing (MSM) unit of the CSIR. These projects include the generation of micro emulsions for advanced particle production, research into components to be integrated into an HIV/Aids diagnostic device, the development of a hand-held diagnostic device to be utilised for water and other health-related diagnostic applications and the development of conducting polymers for fuel cell applications. Each of these projects will be briefly described and the operational and other specific advantages of microfluidic systems for each area will be provided. Finally, the establishment of a microfluidics network in South Africa will be described. This is aimed at allowing interested parties to communicate easily with one another and includes the sharing of information and organization of specialised conferences and collaborations. 2. Advanced emulsion generation Microfluidic methods offer capabilities for precise manipulation and control of fluidic interfaces for various applications, such as the production of emulsions (Engl et al., 2007; Garstecki et al., 2005; Dendukuri et al., 2005; Thorsen et al., 2001). Emulsions are produced by dispersing droplets of one liquid in another continuous liquid phase and are important for use in the pharmaceutical, food and chemical industries,

among others. The monodispersity or small size distribution of droplets in emulsions is crucial in these applications. Advances in microtechnologies and microfluidics have made it possible to accurately control droplet size and create monodisperse emulsions on a micro scale (Shah et al., 2008; Joanicot and Ajdari, 2005; Anna, 2003; Utada et al., 2005; Nisisako, 2005; Kobayashi, 2007; Zhou, 2006).

Various geometries are used for generating emulsions and the most common and simple geometries are the T-junction (Figure 1a) and the flow focussing (Figure 1b). Microfluidic devices, which are based on both of these geometries, have been developed by the CSIR for the generation of emulsions. The microfluidic devices used for generating emulsions are also designed to perform other functions, such as mixing, chemical reaction and particle sorting. Figure 2 shows an advanced microfluidic circuit made from PDMS, where three water-based fluid streams containing two reagents were mixed and then emulsified in the oil phase. The aqueous phases to be mixed were brought together by hydraulic flow focussing before the droplets contacting both aqueous phase A and aqueous phase B were formed. The reagents in the two phases continued mixing inside the droplet through convection and diffusion. The oil phase, which was composed of hydrocarbon oil and a surfactant, suspended and transported the droplets downstream through the channel for storage or further processing. The surfactant increased the kinetic stability of droplets and prevented the droplets from coalescing. The droplet size of the emulsions produced in this manner can be controlled from several hundred microns to a few microns in diameter.

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Numerical modelling and simulation of the two-phase flows has been performed using computational fluid dynamics (CFD). Commercially available CFD software programs have been used as tools to achieve the results. Modelling and simulation also allows for innovation as future designs can be developed and evaluated without being limited by existing experimental testing systems. Droplet formations in T-junction and flow focussing (Figure 1) configurations have been simulated using mathematical code based on the level set method (LSM). In these models interfacial tension, contact angles and fluid-fluid interfaces are important boundary conditions, in addition to the ‘regular’ flow boundary conditions.

(a) (b) Figure 1: Snapshot of models for droplet formation in (a) T-junction and (b) flow focussing devices.

Figure 2: Photograph of emulsion generation in a flow focussing device. The aqueous phases continue mixing inside the droplets. The black lines were drawn to emphasise the walls of the channels.

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3. Components for HIV/Aids diagnostic devices

The University of the Witwatersrand (Wits) and the CSIR are jointly working on a project to miniaturise a component of a unique process, developed by Wits, to detect HIV.

Conventional processes utilise the polymerase chain reaction (PCR) technique to amplify nucleic acid (DNA or RNA). This enables small amounts of nucleic acid, such as viral-RNA, to be amplified and detected with existing technologies. In PCR processes, thermal cycling is required to accomplish nucleic acid amplification, in which three different temperature zones are utilised. However, the

method developed by Wits is performed at a single temperature, providing an isothermal process that is simpler to realise on a micro scale device.

The application of this process is focussed on HIV detection.

Current methods of HIV detection are laboratory-based, take at least eight hours to generate a result and are based on temperature cycling PCR.

This is where the primary novelty of the Wits nucleic acid processes features: it amplifies HIV viral nucleic acid by isothermal amplification that can potentially be completed within one hour.

The viral load is the highest in the window period (before the body starts making antibodies), thus making early detection of HIV possible. Commercialising this method of diagnosis, by means of miniaturisation, has the potential to create a significant impact on the current HIV diagnostic status in South Africa. However, this process of providing HIV viral nucleic acid detection and quantification will rely on three major processes: viral RNA extraction, viral amplification and target detection for quantification. Currently Wits has a method for viral amplification and are working on a Peltier heating system to control the isothermal temperature, but all other processes still need development and integration. The role of the CSIR is to assist in miniaturising this process, including designing and manufacturing the devices and performing microfluidic testing and optimisation. A number of structures

and components have been investigated, including valves, storage wells, fluid transport and fluidic mixing. Software to control the operation of the device has also been developed. The various components have been integrated into a single device and this device is currently being tested and optimised for integration into the full detection device.

It is hoped that microfluidics will provide an inexpensive, reliable and portable HIV detection integrated system that will benefit point-of-care HIV diagnosis and monitoring, where access to conventional testing techniques is not always readily available. The successful miniaturisation of the novel HIV detection process will thus provide an invaluable tool in the HIV diagnostics and monitoring toolkit.

This project receives direct funding from the CSIR and is being performed as part of an MSc research project at Wits. It is hoped that additional funding will be realised to scale up the technical work on the microfluidics project.

4. Smart diagnostic systems (SDS) Since the recent introduction of microfluidic and so-called lab-on-chip (LOC) devices, the most important potential areas of application have been identified as those which would lead to the improvement of quality of life of people in developing countries. This has largely been due to the benefits associated with these devices, namely low cost, ease of use and rapid analysis, in addition to the benefits already mentioned (Chin et al., 2007).

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In addition to the microfluidic design and manufacture, point-of-care (POC) devices would require an instrument which would be capable of reading and analysing data obtained from the microfluidic device. These POC devices are central in the emerging market as devices begin to move from the laboratory to the bed-side. The ability to make medical instruments smaller, portable and lower in cost appeals directly to low resource areas where the need for regular, timely (and often critical) diagnostic feedback far surpasses the availability of qualified staff and expensive lab equipment. A dire need for an affordable POC device that enables the monitoring and feedback of a patient's viral load (when infected with HIV/AIDS) is apparent, now more than ever. It is estimated that there are around 33,000,000 people living with HIV/AIDS across the globe, with the vast majority of this number living in low resource areas such as Sub-Saharan Africa. Introducing a POC instrument which can accurately sample and monitor an individual's viral load count in response to antiretroviral drugs is a very good indicator of the effectiveness (or ineffectiveness) of the treatment. The microfluidic development of such a device has been described in section 3. Another potential application for such a portable device would be for water quality monitoring. Commercial equipment and testing procedures exist for all of the applications mentioned. However, and this is critical, this equipment has inherent flaws which makes adaptability to resource limited settings very difficult, or even impossible. A number of constraints exist where testing is required in low-resourced areas, namely:

• a general lack of electricity; • a need for low cost equipment and disposables (device and reagents); • very few skilled and trained end-users; and • a need for the device to be robust and capable of operating in harsh conditions (e.g. large

variations in temperature and humidity) (Chin, 2009; Yager et al., 2006). It is clear from the above that a radical change in thinking, device design and implementation is required to address these areas.

In order to address some of these issues, the Micromanufacturing and Mechatronics Research Groups at the CSIR have teamed up to research and develop a hand-held diagnostic device that will find application in water quality and health diagnostic areas. The hand-held diagnostic device will combine microfluidics, optics, electronics and software to integrate the necessary components for the complete functioning of the diagnostic system as a portable, stand-alone device. The project has started with the design of a laboratory-based micro flow cytometer, which integrates a number of the components that would be required in a final device (Ateya et al., 2008). This includes the microfluidics, fibre-delivered light source, avalanche photo diode detector for fluorescent detection, collection and delivery optics, and signal processing software. A parallel study is looking at the best platform for the implementation of a hand-held device. This will be the first proof of concept of a device that can to detect the very low levels of fluorescence emitted from excited biological samples. Figure 3 shows schematically the experimental setup for such a device. In Figure 3, (2) indicates the fluid of interest containing the fluorescing particles. Fluid (1) is split into two channels which force a sheathed flow in order to constrain the particles to pass the excitation/detector in a single file. The input channel (3) for the laser diode excitation source (6) and input fibre (7) are shown. The detection channels are indicated by (4), and are utilised either for multiple detection points or for detection of different wavelengths, with (8) being the detector and (9) the output fibre. The computer (10) is utilised for microfluidic control and signal analysis of the output signal. The interrogation area (11) is shown. The fluid output is shown by (5), and this can lead to sorting or storage of the particles based on the signal analysis and determination of the particle characteristics.

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Figure 3: Schematic layout of the flow cytometry device. 5. Micro fuel cell There has been a significant increase in demand for renewable energy sources in recent times. This has lead to intensive research on fuel cells worldwide. The emergence of micro fuel cells as power sources for technologies such as laptops, personal digital assistants (PDAs), cell phones and other mobile devices, has lead to micro fuel cells receiving much research attention and funding. Most micro fuel cells are Direct Methanol Fuel Cells (DMFC) fuelled by methanol because of their room temperature operation and easy fuel storage (Wozniak et al., 2004; Zhong et al., 2008; Ji and Lou, 2008). A typical fuel cell consists of a MEA (Membrane Electrode Assembly), flow field plates, electrode plates, end plates and sealing gaskets. These components are shown in Figure 4. The MEA is not visible, but it is sandwiched between the two graphite flow field plates. In terms of cost, mass and ease of assembly, it would be advantageous for many of these components to be integrated into a single component. In this regard, a project is under way to combine the sealing gasket, flow field plates and electrode plates into a single component. Polymers such as polydimethylsiloxane (PDMS) become suitable structural candidates for this fuel cell fabrication due to their light weight, good mechanical and anti-corrosion properties, and their ability to act as sealing gaskets and to be moulded. Unfortunately, their electrical conductivity is typically very low. The current project looks at utilising carbon black, silver powder and carbon nanotubes as filler material for PDMS-based composites in order to improve the electrical conductivity of the PDMS composite. The introduction of such composites as structural material will reduce the mass and the size of the micro fuel cell and therefore optimise the current micro fuel cell models. Research is focussed on the customisability and optimisation of thermal, mechanical and electrical properties of the conducting PDMS composite.

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In addition, the project also considers low-cost manufacturing techniques for the manufacture of certain of the fuel cell components. Figure 5 shows a laser cut PDMS flow field plate. This method of manufacture is ideal for rapid testing of the device as no moulds are required.

Figure 4: Miniature fuel cell developed at the CSIR. 1. Rubber sealing gasket, 2. Graphite flow field plates, 3. Copper electrode, and 4. Laser-cut perspex (PMMA) clamping plates.

Figure 5: Laser-cut PDMS fuel cell plate.

6. Facilities

For the above-mentioned microfluidic research areas to be realised, a number of facilities are available at MSM, CSIR. Figure 6 shows the clean room, where SU-8 structures can be photolithographically manufactured onto silicon substrates. For this purpose the clean room contains a spin coater, two hotplates, a mask aligner and a fume hood for structure development. The clean room process is shown schematically in Figure 7a. Figure 7b shows the process of casting PDMS structures from the mould made in the clean room. The basic process is as follows (note that spin coat, baking and exposure parameters are dependent on the required photoresist thickness – values given are for layers with a thickness of 150 µm): 1. Clean silicon wafers in acetone in ultrasonic bath for 15 minutes. Dry with nitrogen (Figure 7a.1). 2. Oven bake at 2000

3. Spin coat SU-8 negative photo resist onto a 4 inch silicon substrate (spin speed 1850 rpm, SU-8 2100) (Figure 7a.2).

C for 30 minutes.

4. Soft bake for five minutes at 650C and 30 minutes at 950

5. Expose with mask aligner (i-line 365 nm, 260 mJ/cmC.

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6. Post-exposure bake (PEB) for five minutes at 65) (Figure 7a.3).

0C and 12 minutes at 950

7. Develop with SU-8 developer (visual check, approximately 15 minutes) (Figure 7a.4). C.

8. PDMS (silicone elastomer and curing agent mixed in a 10:1 ratio) is poured onto the mould template (Figure 7b.1).

9. Degassing of the PDMS utilising a vacuum pump and desiccator. 10. PDMS cured at 650C for one hour.

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11. PDMS inverse structure peeled off mould (Figure 7b.2). 12. Structure mounted to laser cut Perspex templates and holes punched for fluidic inlets and outlets

(Figure 7b.3).

The laboratory for performing this construction and testing contains a spin coater, oven, oxygen plasma machine, microscopes, syringe pumps and various other pieces of equipment.

Figure 6: Photograph of the completed clean room facility.

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Figure 7: (a) (1-4) Clean room process for the creating of structures on silicon wafers, and (b) (1-3) the manufacturing process for PDMS microfluidic devices.

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7. Microfluidic research network Microfluidics research and development has only recently received attention in South Africa. To the best of the authors’ knowledge, with the notable exception of micro electronics and MEMS (micro electro mechanical systems), there are no universities teaching a micro-manufacturing or microfluidics curriculum. The result is that there are very few people with the knowledge required to design and manufacture microfluidic components and devices. Due to the multidisciplinary nature of the field, it would be ideal to introduce course work in both engineering and science faculties, and for students to be encouraged to do post-graduate studies in these fields. There are currently a number of niche areas of microfluidics and micro-manufacturing which are being pursued by industry, universities and research institutes. Few of these researchers are sharing their knowledge or collaborating, which makes it difficult to form a cohesive microfluidic community of like-minded people. When expertise is required, it is, for the most part, sourced internationally. In order to develop a community with microfluidics and micro-manufacturing as the central focus point, the CSIR has launched a microfluidic research network. A website (www.microfluidics.co.za) has been set up where people can indicate their interest in the development of such a network and can share ideas for its further improvement. This will encourage communication between the various parties. Once interest has been ascertained, it is envisaged to hold regular meetings and workshops to discuss projects, collaborate, exchange ideas and generally get to know the various people and institutes interested in microfluidic development.

8. Conclusions Micro-manufacturing and microfluidics are critical emerging research areas and will continue to receive considerable research focus. One of the main reasons for this is the considerable impact these technologies are expected to have on global health issues. It is thus imperative that this area of research receives funding in South Africa. The CSIR has taken the lead in establishing facilities in order to start researching this area. A number of projects are being actively researched. A start has been made to develop human capital in the area of microfluidics. However, additional support is required to further develop human capital in this area and o continue to develop the facilities that have been established. 9. Acknowledgements This work was supported by funding from the following sources: The CSIR Executive: Establishment of the clean room facility. The CSIR parliamentary grant funding: Microfluidics and micro fuel cell projects. Advanced Manufacturing Technology Strategy (AMTS): Development of a micro manufacturing facility and advanced emulsion systems project. We gratefully acknowledge this funding.

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10. References Anna, S.L., Bontoux, N. and Stone, H.A. 2003. Formation of dispersions using flow focussing in microchannels, App. Phy. Lett, vol 82, no. 3, 364-366. Ateya, D., Erickson, J., Howell Jr, P., Hilliard, L., Golden, J. and Ligler, F. 2008. The good, the bad, and the tiny: a review of microflow cytometry, Anal Bioanal Chem, vol 391, 1485-1498. Chin, C.D. 2009. Biotechnology for Global Health: Solutions for the Developing World, Consilience, vol 1, no. 1. Chin, C.D., Linder, V. and Sia, S.K. 2007. Lab-on-a-chip devices for global health: Past studies and future opportunities, Lab on a Chip, vol 7, 41 – 57. Dendukuri, D., Tsoi, K.T., Hatton, A. and Doyle, P.S. 2005. Controlled Synthesis of Nonspherical microparticles using microfluidics, Langmuir, vol 10, 2113-2116. Engl, W., Backov, R. and Panizza, P. 2007. Controlled production of emulsions and particles milli and microfluidic techniques, Curr. Opin. Colloid Interface Sci. Erickson, D. and Li, D. 2004. Integrated Microfluidic Devices, Analytica Chimica Acta, vol 507, 11-26. Garstecki, P., Ganan-Calvo, A.M. and Whitesides, G.M . 2005. Formation of bubbles and droplets in microfluidic systems, Bulletin Polish. Academy. Tech Sci., vol 53, no. 4, 361-372. Ji, X. and Lou, W. 2008. Design of a new direct methanol fuel cell utilizing MEMS technology, WSEAS Transactions on Power Systems, vol 3, no. 4, 172-181. Joanicot, M. and Ajdari, A. 2005. Droplet control for microfluidics, Science, vol 309, 887-888. Kobayashi, I., Uemura, K. and Nakajima, M. 2007. Formulation of monodisperse emulsions using submicron-channel arrays, Colloids and interfaces: Physicochem Eng. Aspects, vol 296, 285-289. Nisisako, T., Okushima, S. and Torii, T. 2005. Controlled formulation of monodisperse double emulsions in multiple-phase microfluidic system, Soft Matter, vol 1, 23-27. Shah R.K., Shum, C.H., Rowatt, A.C., Lee, D. et al. 2008. Designer emulsions using microfluidics, Mat. Today, vol 11, no. 4, 18-27.

Sia, S. and Whitesides, G. 2003. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies, Electrophoresis, vol 23, 3563-3576. Thorsen, T., Roberts, R.W., Arnold, F.H. and Quake, S.R. 2001. Dynamic Pattern Formation in a Vesicle-Generating Microfluidic Device, Phys. Rev. Lett., vol 86, no. 18, 4163-4166. Utada, A.S., Lorencaueu, E., Link, D.R., Kaplan, P.D., Stone, H.A. and Weitz, D.A. 2005. Monodisperse double emulsions generated from a microcapillary device, Science, vol 308, 357-541.

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Wozniak, K., Johansson, D., Bring, M., Sanz-Velasco, A. and Enoksson, P. 2004. A micro direct methanol fuel cell demonstrator, Jounal of Micromechanics and Microengineering, vol 14, S59-S63. Yager, P., Edwards, T., Fu, E., Helton, K., Nelson, K., Tam, M.R. and Weigl, B.H. 2006. Microfluidic diagnostic technologies for global public health, Nature, vol 442, no. 7101, 412-418. Zhong, L., Wang, X. Jiang, Y., Zhang, Q., Qiu, X., Zhou, Y. and Liu, L. 2008. A micro-direct methanol fule cell stack with optimized design and microfabrication, Sensors and Actuators A, vol 143, 70-76. Zhou, C., Yue, P. and Feng, J. 2006. Formation of simple and compound drops in microfluidic devices, Phys. Fluids, vol 18, 1-14.