7.1 Application Fields

Massively parallel screening methods that apply combinatorial (bio)chemistry in robotic-assisted fluid handling systems is one approach to handling extremely small samples. Microarray technology in medical diagnostics is another example of the implementation of experimental screening, which benefits tremendously from microfabrication techniques originating from the microelectronics industry. Expanding capabilities in the life sciences, which studies biological material, however, requires new solutions for chip manufacture. A variety of miniaturized, liquid-handling devices employ the principle of standardized microtiter plate formats. The miniature reaction vials are organized as an x–y matrix, which allows fast scanning across each of the reaction chambers. Flow-through devices allow continuous analysis on chip applying, for example, capillary electrophoresis (CE), chromatography or non-destructive optical techniques, all of which are integrated into one hand-held measurement system.

Continuing innovations in genomics and in analytical technology, provide pharmaceutical manufacturing and biotechnology businesses, such as brewing or food, it is hoped that these developments will benefit from new microfluidic methods. The concept of carrying out chemistry in confinement can allow the rapid development of new drugs and pharmaceutical treatments, as well as green and healthy food products.

Many innovations in these fields can be traced back to a basic phenomenon in fluid dynamics: the surface tension of incompressible fluids. A pressure is required to drive liquid through a capillary (microfluidic channel). The understanding of how different forces inside of capillaries interact such that they can be used effectively to transport compounds in these novel systems is an important area of research. Here, we will consider some working principles from a practical point of view, with the intention of being able to adapt a variety of operations known at the macroscale: pumping, dispensing, mixing, quenching, etc. Microfluidics can also be operated in combination with array strategies for feeding different samples to the chip, e.g., coupled with a mass-spectrometer. Many established conferences and peer-reviewed journals (Analytical Chemistry, Electrophoresis, Sensors and Actuators, Journal of Microelectromechanical Systems, Transducers, μTAS) have turned their attention to cell manipulation and analysis within artificial microenvironments. These arrangements allow well-defined spatial resolution for the observation of biological processes inside and in the extracellular matrix of living cells.

(Bio)chemical processes are investigated as part of the in vitro and in vivo testing of pharmaceutics, using animal models and volunteer human subjects. Pharmaceuticals may behave differently in vivo to what may be expected from in vivo studies; drugs may be unexpected effective against other diseases but can also cause undesired side effects. Knowledge of the overall (complex) behavior of a (bio) pharmaceutically active substance is difficult to generalize and statistically significant amounts of data have to be collected. Despite all the technological know-how, the risk of mistreating an individual is still high. Drugs can cause the agent of a disease to mutate, or cause undesirable changes in human genes (phenotype). Genotype and phenotype testing have become available for the diagnosis and evaluation of HIV drug resistance, and, in theory, resistance assays can be used to determine which drugs will be effective against an individual’s strain of HIV, so that the appropriate treatment can be selected. There is still, however, much controversy about the accuracy of these diagnostic tools, and their usefulness as predictors of treatment success. Microfluidic-assisted systems for cell biology can help to elucidate these mechanisms.

Microfabricated devices can address cells, cell components and environmental features in the conglomerate of cultured and in vivo bio-environments, which will allow researchers to follow the dynamics of an individual cellular and molecular control mechanism in detail. This understanding of cell regulatory processes may be utilized to reconstruct even more complex bioprocesses including, for example, neuron circuitry, and bone and tissue repair mechanisms. These investigations may also identify an appropriate response to overcoming the risk of ill-treatment in case of pathogen mutation. Microfluidic devices can furthermore assist the biomedical sciences in general. This hypothesis concerns the controlled operation of moving, distributing and storing reagents and samples resulting in so-called cell culture chips with integrated analytical functions, e.g., utilizing the aforementioned integration concept of electrochemical and optical sensor technology. Miniaturized sensors are capable of interacting with cells at the same length scale, which may offer additional information.

The flow-through concept has several advantages over a batch-type approach, although both aim for high throughput solutions, massive parallelism and high reliability. Batch processes, using multiple pipettes, are prone to contamination, cross-interference, and are slower and less accurate in dynamic response when compared to the microfluidic approach, with its spatial discrete reaction and detection features. Additionally, titer plates are difficult to control due to evaporation, but if covered vials are used, fast robotic access will be limited. The polymerase chain reaction (PCR) “on chip” is a typical example of enhanced operation using a flow-through microfluidic device for finger-printing in cell biology. Microfluidics are also preferred for basic research in the life sciences, molecular diagnostics, pharmacogenomic profiling for personalized therapeutic planning, and the detection of food contaminants and bio-weapons. At present, titer plates not only serve as convenient sample or reagent storage trays, but additional functions are increasingly being integrated into the same platform. This will also allow rapid data correlation from 96 (384,.. etc.) analysis positions in a single run. The cavities of a titer plate can also serve as a reaction vessel or cell culture chamber, if they are nourished by a microfluidic network and allowing for cross-culture interactions. These advances in titer plate development, show the clear movement towards integrated microfluidics, which makes it a next logical step to pursue this trend and go for massively parallel flow-through microsystems for screening. Microfluidic on-chip processes are expected to improve the control of the reaction sites due to optimized heat and mass transfer. However, method development for Lab-on-a-Chip is still slow, and is particularly hampered by the high investment costs for customized chip technologies compared to the already well-established batch-based array-type assays in biology. Categorizing the various fluidic layouts into functional subunits, which can be assembled in a similar way to electronic design layout for standard components and better methods for in silico modeling, however, should make-up or this relatively slow progress in the near term future.

Advanced on-chip fluid electrokinetics will allow samples and other reagents to be prepared by more sensitive methods, and will also allow multiple measurements to be made from the same sample. Sample clean-up and enrichment prior to capillary electrophoresis has been studied earlier by conventional systems, for example, by Busher and co-workers [66]. These techniques, including selective extraction methods, are very suitable for microfabricated analytical systems, and it will be of great interest to develop integration strategies for microfluidics devices, often referred to in the MEMS community as micro-total analysis systems (μTAS), because of their original implementation as an analytical tool [1–3].

This chapter will consider the conceptual development of the lithium sensor as an example. I have been fascinated by the development of this device myself, probably to a greater extent than was strictly relevant. Innovation in industry is seeded by research projects. A good example of this is the LICETAS (Lithium Capillary Electrophoresis Total Analysis System), conducted in the BIOS-Lab-on-a-Chip group, headed by Prof. Dr. Ir. Albert van den Berg, at the University of Twente and financially supported by the Dutch Science and Technology Foundation (STW). Consideration of the LICETAS project may help us to answer an important question: How should governments and industry leaders make educated choices over which research should be funded, when all the suggested research projects are, of course, good ideas in the first place? LICETAS did not appear to be exceptional among the many excellent ideas presented by scientists, and was still some way from clinical application [4]. Recently the device has been further developed by Medimate BV, The Netherlands, and reported by Yager as a cutting-edge development for point-of-care diagnostics citing Floris et al. in LabChip [5, 6].

Clarifying the key differentiators versus the incremental progression of a scientific contribution may help in making educated decisions for funding research, either by government evaluators, or by industrial organizations looking for applications for their products. However, I can only let the section in this chapter speak for itself regarding whether the time and the money were well spent in turning the measurement of the conductivity of a liquid into a feasible concept for clinical point-of-care applications. In contrast to the many good books available in this field, I add my own personal experience of post-doctoral research. I hope to inspire readers to add their own knowledge to develop new micro- or nanofabricated products. Maybe there is no longer that much room at the bottom for miniaturization technology, however, focusing on the More than Moore principle in the applied sciences may successfully pave the way to an uncrowded business niche in industry [7, 8].

7.2 Microfluidic Components

Generally, it is possible to bring a sensor to the measurement point, and simply expose the sensor to the environment of the measurand, but often this is not considered to be a practical solution. Particularly if the sample to be measured is blood, an off-line measurement is the method of choice. Also for many chemical engineering systems the decoupling of sample and production lines is often preferred, because the sample is crude and contains many more compounds than just the measurand. The sample needs to be worked-up, and sample preparation methods are needed if a measurement is to be conducted without interference from other compounds. Microfluidic components, and in particular a microvalve, could optimize sample preparation techniques, particularly when very small sample volumes are available, only. We therefore need to understand the control of transport phenomena of minute amounts of fluids (gases or liquids) when they are confined and moving from place to place. The efficiency of this transport is a particular concern in microfluidic systems. Most of the strategies using a variety of pumping mechanisms are inefficient at the small scale. On the other hand, at the very small length scale nature provides us with the phenomenon of diffusion as an efficient means of transport. Nevertheless, microscale operations, pumps, valves and mixers are still important structural elements for many microdevice operations. For details of the theoretical background of fluid mechanics, the study of continuum fluid dynamics equations and the laminar fluid flow patterns in confined spaces is important. With respect to the latter, I would like to refer to the physico-chemical background literature. An initial starting point for advanced reading is the compilation of publications from 2006 in the special issue on “Lab on a Chip” in Nature [9].

Microfluidic systems may be designed using any materials available in the microfabrication tool box: semiconductor materials, glasses, quartz, plastics, ceramics and even metals. With respect to valving and pumping, dynamic structural control is important. Examples do exist that use chemical interference on the valving or pumping structure, e.g., applying pH-induced volumetric changes in the case of elements fabricated from porous hydrogel materials, but mechanically rigid, structural mechanisms are still preferred in valve and pump designs. This is probably because they have a faster reaction time and more accurate mechanical placement. Prior to studying microfluidics it is strongly advised that the reader gains an overview of basic fluid mechanics at the macro- and mesoscale.

This book will introduce examples of microfabricated components for fluid handling, and the handling of the solutes in those fluids at the microscale in the next subsection. Some examples of non-moving structural elements that explore diffusion phenomena are also discussed. I anticipate that once some examples of the need for fluidic devices at this scale are illustrated, then the advanced study of transport and interface phenomena becomes much more exciting, and this study of illustrated practical cases will stimulate their industrial applicability.

A very good overview of the trend in micropumps, microvalves and micromixers has been presented within the context of the development of polymerase chain reaction (PCR) chips by Zhang et al. [10]. Specifically with respect to microvalves, Oh and Ahn presented a review article in 2006 [11]. Two years earlier, Laser and Santiago presented a review on micropumps [12]. Recently, these reviews were followed by an overview on pneumatic and hydraulic microactuators by De Volder and Reynaerts [13]. The latter discuss the importance of early MEMS research and development to microfluidics applications and illustrate a variety of designs in a comprehensive overview. Readers with a larger appetite for the design and application of fluid microactuation will doubtless highly appreciate the guidance on pneumatic and hydraulic microactuators given by De Volder and Raynaerts’ review.

7.3 Controlled Transport by Diffusion

Controlling chemical operations in a microfluidic chip may be achieved solely by diffusion. The laminar flow regime supports the operation of two streams merging from two different inlet channels into one channel, while the flow remains discretely side-by-side inside of this one channel. Solutes from one stream can now enter the other, and in the interface chemical reactions may occur. This exchange of solutes may be utilized for gradient dosing or extraction purposes. The exchange between the two streams is entirely limited by diffusion. Figure 7.4 demonstrates a simple H- and T-shaped microfluidic layout which can be produced by microfabrication in glass, or special plastics supporting optical transparency, and thus visualization of the events occurring in the device. An even simpler version may be designed from wicking paper. This was recently highlighted as a potential ultra-low cost solution for global public health care by Whitesides and Yager [20, 21]. By means of these types of structures a variety of applications have been demonstrated, some of which have also been commercialized as single-use medical diagnostic devices such as, for example, the H-Filter^® and T-sensor^® Access™ cards offered by Micronics, Inc., USA [22].

7.4 Integration for Microfluidic Transport, Sensing and Dispensing

In standard (bio)chemical handling, fluids containing analytes or reagents are transferred by means of a pipette. This may be an automatic pipette, with multiple tips loaded/deloaded in parallel, but single-tip pipettes are still in use, particularly in research environments. Using microfluidic transport for a similar operation in an experimental set-up on chip requires some level of component integration. The capillary effect, related to the above mentioned surface tension of the fluid and the size of the microchannel, could also be used. Hereby important parameters are the filling length of the channel h against the ambient air pressure at the end of the capillary proportional to the surface tension γ, the specific weight of the fluid w, the angle between the edge of the free fluid surface θ and the channel wall, and the radius a of the capillary tube. This basic concept is cleverly exploited in the diffusional set-up given in Figure 7.4. To eventually allow the lock-in handling of liquid dispensing after loading, a diverse range of integrated devices have been designed and run in an automatic, or at least semi-automatic fashion. A pumping action can be produced by other means besides simply pressurizing the inlet of a channel through a piston-driven, syringe pump. The application of an electrical potential across the capillary length will also result in a pumping action. This technique applies a field of up to a few 100 V/cm and is called electroosmotic fluid flow. The method appears to be easy, but it still requires some effort to miniaturize all the components, particularly the high-voltage power supply.

Leak-tide integration of electrodes has also demanded the attention of manufacturers in the field. Micronit Microfluidics BV, Enschede, The Netherlands, for example, has tackled the challenges of robust, integrated manufacturing in glass, and today offers a range of standard and customized microfluidic glass chips. Figure 7.5 illustrates these examples of product implementation in this novel, life-science oriented, microfabrication service industry.

7.5 Lab-on-a-Chip

The Lab-on-a-Chip originates from the initial development of miniaturized sensors and fluidic handling devices within one miniaturized package, the chip, so-called micro total analysis systems (μTAS) made up the first. A much broader implementation of these techniques quickly followed. There was considerable impetus for scientists already working on miniaturization to investigate means of measuring chemical reactions because of the large number of chemicals that are used in fabrication processes. While the latter not only attracted more and more scientists with a chemistry background to the research area of miniaturization technology, it also motivated the discovery of efficiency enhancement of chemical reactions performed in microfluidic channels.

Originally, Lab-on-a-Chip technology focused on analytical devices, and established such concepts as flow injection analysis (FIA) on chip. Throughout the last decade, however, we have experienced a revolution in this field in the life sciences. These have targeted a wide range of (bio)chemical reactions in cell cultures and clinical diagnostic point-of-care devices. The driver for this is the emergence of genetics as a medical field, where conventional analytical power is insufficient to sustain the required rate of experimental progress. So-called sample-in-answer-out capabilities are of considerable interest in life sciences [25–27].

Generally, micro- and nano-scale chemistry uses the increased surface-to-volume ratio resulting from the implementation of microfluidic devices. Combing μTAS and chemical synthesis research has led to the broader definition of this technical field, which is now known as Lab-on-a-Chip technology. The current prototypes translate the working principles of many standard laboratory analysis and synthesis techniques onto a chip card format (like a credit card). By fitting chemical reagents, test tubes, flasks and stirrers, pumps and valves onto a footprint hardly larger than a postage stamp has realized a paradigm shift in miniaturization. Figure 7.6 gives an introductory overview to the extensive developments of this novel field of technology.

This on-going analytical revolution started with an extremely steep development curve in the 1980s, which approximately 30 years later, is reaching saturation of early curiosity-driven research. Although one cannot talk about a specific killer application this saturation clearly pays-off from a commercial point of view. The field of microfluidics controls the transport of gases and liquids inside the chip by a variety of structural components, as discussed in the previous section. Investigations of novel strategies for moving fluids in a channel network is described extensively in the literature. The initial developments in microfluidics were an off-spring of MEMS technology. These developments integrated valves and pumps for flow rate measurements, and mass sensors for highly defined gas supplies and safety equipment in the semiconductor industry. They also gave rise to pumps for controlled drug delivery for the chronically ill. Microfluidic circuits were also explored as far back as the 1970s in logic devices, because of their insensitivity to electromagnetic radiation.

Nowadays, microchemical reactions on a chip are popular as analytical systems for the same reasons as in analytical chemistry, and they are therefore branded as their own branch of Lab-on-Chip research and development, known as microreaction science and technology. Early on-line measurement systems were considered to be inferior to the possibilities of a well-equipped laboratory. Eventually, the introduction of flow-injection analysis, utilizing miniaturized systems was an economically driven choice. When it is more important in a bio(chemical) production line to have a rapid, crude measurement of product composition, miniaturization supported the rapid adoption of such μ-FIAs to be integrated into the production processes. The success of this supported the further validation of miniaturized sensor systems in field operations. This created a demand for the development of commercial microflow injection analysis (FIA) systems.

A FIA for (bio)chemical process lines is constructed as a mass flow controller for gases, in which a bypass line is utilized to carry out the measurement. One very successful example of chemical in-line measurement is the miniaturized gas chromatograph, marketed by Thermo-Fisher, who acquired the start-up company C2V BV. C2V developed this measurement system from concept to volume manufacturing.

Once the μ-FIA concept was introduced, researchers started to explore other application fields and to translate the micro total analysis approach to a variety of measurement challenges, including biology. The idea that biology could benefit from a system-in-a-card format is not all that obvious, since very sophisticated cell handling instrumentations already existed. Nevertheless, chips were introduced and tested, promoting the concept of automating cell manipulations at the level of a single cell. Microfluidic chip systems may actually be used to manipulate many thousands of single cells in parallel by integrating the capturing, triggering and analysis functions all into one system. Consideration had to be taken of the relevance to biology of taking measurements at the level of just one cell, since studies at the level of a population are usually necessary. Specifically, chips were considered as a means of learning more about cells in suspensions. Initial chip layouts utilized microfabrication to demonstrate miniaturized bio-handling systems similar to the operations that would normally be performed in an established fluorescent-activated cell sorter (FACS). FACS scanning the individual properties of the cells that previously had the opportunity for cell–cell interaction within the tissue population is a powerful tool for deriving statistically meaningful bio-information. Established non-chip formats allow a large number of cells to be counted by appearance (scatter, fluorescence from marker uptake, etc.), but without the possibility of tracing an individual cell in time throughout the experiment. If the single cell approach on chip is relevant biologically speaking then this design may be supported. However, the design of experiment with single cells remains a matter of further debate. The fact that in-vitro cultures of certain cell lines, in which cells are also isolated from their natural environment, are also a popular tool in biological experiments suggests that information collected from population, single-cell or tissue level can only model the in-vivo situation up to a certain level, and all the findings together may not produce a full understanding of real-life systems. We will not debate the various models for bio-medical experiments here, but will introduce the lab-on-a-chip concept and its technological realization by microfabrication as a potential approach for biological studies. Figure 7.7 shows the first microchip-based cytometer fabricated and tested by Micronics [31]. It clearly has been a milestone in the development of microfluidics for cell research.

The following subsections present an overview of microfluidics devices for particle and cell sorting, and miniaturized cell culturing systems, with a variety of integrated functionality. Micronics microfluidic card technology is based on laser micromachining, or stamping of foils and subsequent assembly by lamination.

7.6 Device-to-World Connections: The MATAS Concept

In Chapter 6 we discussed a set of sensors used in microfluidic devices. Microfluidics applications aim for hybrid integration in a similar way to the concept of combining different micromechanical and electronics sensors into one measurement system. The implementation of different devices and components into one platform is often referred to as the Lab-on-a-Chip concept, illustrated in Figure 7.6. Today, such systems have found their way from universities into chemical analysis, separation technology and synthesis (microreaction technology). The health-care market demands faster and faster response on differentiating disease patterns, and therefore a biolab infrastructure now demands much more flexible processes and rapid updates of protocols for faster screening.

Although conventional mechatronic robotics still competes with the revolutionizing possibilities of microfluidics, the latter is now widely recognized in the life sciences and industrial (fine-chemical) process control markets. The benefits of miniaturization in fluid handling environments are the reduced consumption of reagents, shorter temperature cycling times, faster mixing and a high degree of automation. These factors can result in a higher throughput, and a reduction in costs compared to conventional methods. One unique technology that is suitable to deliver a flexible and cost-effective realization of a Lab-on-a-Chip system is the MATAS (Modular Assembly for (micro) Total Analysis Systems), produced by LioniX BV, Enschede, The Netherlands [35].

MATAS is a highly standardized, modular technology for the design and fabrication of Lab-on-a-Chip systems-in-a-package. The technology enables the hybrid integration of microfluidic components, micro-optical devices and control electronics into one platform. This modular microfluidics kit has been selected for on-line measurement of pH and conductivity, and merges precision engineering with silicon micromachining. Figure 7.8 shows different components and devices of the MATAS.

Systems made up of these types of modules have so far been used as microreactors, sensors for pH, conductivity and ion concentrations, CE-chips, electrodes, flow sensors, micropumps, optical detectors, filters, valves and pressure sensors. All these different modules can be tested individually and exchanged just as quickly as ICs and resistors on a printed circuit board (PCB). The MATAS concept, originally developed by University of Twente’s spin-off 3T, of which the microfluidic division was acquired by LioniX, allows the user to integrate hybrids of different components into one customized package. Modules can include optics-based sensors, actuators and microelectronics which are already manufactured in large-scale production lines, as well as structures produced by speciality foundries. Figure 7.9 depicts a fully assembled μTAS for the detection of pH and ammonia.

7.7 From the Lab Bench to Industry: Microchip Capillary Electrophoresis

Capillary electrophoresis (CE) is a well established separation method in analytical chemistry. At the conventional scale small bore tubes must be used, in order to quickly dissipate the heat that is generated by the current flowing through the capillary at the applied high electrical field strength (several tens of V/cm). Under these conditions, the ions within the sample will separate, since they diffuse at different rates within the analytical medium. The separation length of a conventional system is approximately one meter because conventional fluid handling systems cannot produce the tightly defined picoliter-sized plugs that microfluidic-implemented systems can. Figure 7.10 depicts an example of a conventionally operated CE system, together with a chip-based CE apparatus with a total foot-print of a desktop computer, as developed by Shimadzu.

I was involved in LICETAS, a governmentally funded project, from 2001 to 2005 as a post-doctoral researcher together with an analytical chemist, Elwin Vrouwe, who at the time was working for his PhD. Dr. Vrouwe currently works as an industrial researcher at Micronit Microfluidics BV and I decided to continue my academic career at University of Twente. Nevertheless, I believe, it may be valuable to discuss some aspects of my involvement with the early efforts of the LICETAS innovation track in the context of this book. The project demonstrates an example of how a small seed-project of 25,000.00 Euros, granted for knowledge transfer to a spin-off company may make all the difference to industrial success.

LICETAS uses microchip electrophoresis with an integrated conductivity sensor, for the measurement of lithium in blood from a finger stick sample. This brings us to the set of subsections that describe the implementation of microfluidics devices at the point-of-care (PoC).

7.8 Conclusions

Original studies of the etching of micromechanical diaphragms for pressure sensors led first to the development of micromechanical actuators, and then to microvalves and pumps in microfluidics. Understanding diffusion and other transport phenomena, of course, is as important in confined environments as understanding the mechanics of pumping. The accurate fabrication of microfluidic channels in glass has opened up the possibility of studying micro- and nanofluidic behavior by advanced optical microscopy. Fluorescence and absorbance methods have been developed to determine the type and concentration of compounds in chemical mixtures. This plethora of novel opportunities has allowed the study of chemical reactions in-situ as well as the manipulation of delicate proteins or cells. Research on the optimization of soft-matter handling protocols is on-going in the field of Lab-on-a-Chip technology.

Studying and utilizing microfluidics has given rise to new applications. Although the microfluidic service industry is already well established, its full potential has yet to be explored. Possible avenues include chip layout services, provision of accessories and chip manufacture on demand. In this chapter, we discussed the potential for a lithium point-of-care microchip device. Estimation of the potential of these types of developments is not easy, and proof-of-principle studies (feasibility) can help to reduce the economic risks. Standards for this type of measurement protocol are yet to be formulated, so patenting is not a real indicator that the solution has an industrial applicability. However, it is applicability that is demanded by the European patent law actually for a patent to be granted. LICETAS was at a very early stage of development when it was transferred to a spin-off company, however, know-how was all the young start-up needed.

Work at the research stage showed the lithium peak appearing within the electropherogram drawn from a blood plasma sample as early as 2002; the method was not validated for its accuracy in a clinical setting. The results therefore were of generic interest to the scientific community, but not of importance to the medical diagnostics market because of the lack of a proof-of-concept for quantitative validation of the method. As soon as the quantitative reliability of the method could be demonstrated, the process of technology transfer to a company made sense. In due course, the required multidisciplinary know-how was collected by the newly formed spin-off company and turned into a novel product with an appropriate patent portfolio to commercially back-up the company’s innovative technology.

Early stage technology transfer is a highly feasible approach to bring innovative solutions from the university to the market, but the LICETAS project operated at an extremely high risk to avoid its results being shelved as soon as the PhD thesis had been defended successfully. The fact that this did not happen was probably based on the right people being present in the right location, at the right time within one dedicated project team, and an appropriate funding agent (the Dutch Science and Technology Foundation, STW) sharing the one vision of bringing technical sciences to the marketplace.

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