DESIGN
Institut für Mikrotechnik Mainz GmbH, Fluidics and Simulation, Mainz, Germany
Closer to the laboratory
Micro-total analysis systems (µTAS) or lab-on-a-chip systems (LoC) allow nonskilled people to perform complete and complex analyses, for example, for diagnostics or the detection of allergens, harmful bacteria and toxic substances. The benefits of these devices include simplified handling, increased reproducibility and reliability, faster analysis time, lower sample and chemical consumption, and lower contamination risk.1,2,3
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Figure 1: Modular system for sample preparation. Proteins are extracted from foodstuff for a quantitative detection.
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Many papers in the field of µTAS/LoC deal only with single separated steps that form part of a whole analysis.4 These steps include cell separation, cell lysis, deoxyribonucleic acid (DNA)/protein purification, metering and mixing, DNA amplification and detection methods. A number of microarray chips for DNA analysis are offered commercially, but fully integrated microanalysis systems are hard to find. No system exists that can claim to replace a laboratory from the very beginning through all the stages of analysis until the final detection. The different elements/modules that can fill this gap to form a fully integrated system are outlined in this article. Figure 1 shows an example of such a system. This system comprises eight modular chips of 6434332 mm3 and the necessary mechanical actuation underneath. This system includes the extraction of proteins from food stuffs, removal of unsolved sample particles, generation of a dilution series and performance of an enzyme linked immunosorbent assay (ELISA) for the quantitative detection of a protein of interest. This method order is the same as that of a traditional assay.
The sample
Typical samples addressed by LoC systems are of human origin such as blood, urine, sputum, tissue samples and smears; and environmental origin such as soil or food. All of these consist of a variety of components, but usually only one of these is of interest, either specific DNA fragments or low concentrated proteins correlated with specific diseases. For example, in blood, millions of erythrocytes can hide the handful of cancer cells being sought;5 or in soil, millions of harmless bacteria may hide the presence of a pathogenic organism.4 Therefore, a purification and/or concentration step at the beginning is essential. Often a starting volume of several millilitres is required, which must be reduced to a few microlitres or less and contain all the targets, all within the microchip.
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Figure 2: Module for the transfer of solid samples into a liquid phase. The vigorous mixing used for macrosamples is replaced by a peristaltic pump.
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As well as the difficulty of taking a representative sample, which is the same for any analysis, the sample must be homogenised and rendered into a liquid. This step is missing in most of today’s microsystems. Typically, these systems are restricted to using liquid samples or analysis must begin with a pretreated sample obtained via a lab-scale method. A solution derived from successful laboratory methods is shown in Figure 2. A solid sample is mixed with an extraction buffer in a flexible tube and kneaded by a peristaltic pump to bring the target protein into solution. The extracted sample is then transferred automatically into a microfluidic system via an integrated microvalve.
Filtration and concentration
The next step after extraction and preconcentration is the removal of undissolved or unwanted sample components. Typically, cells have to be separated from their original matrix to obtain a cell-free liquid such as blood plasma or an extremely small volume of the cells of interest must be obtained. Some methods of removal together with their respective advantages and disadvantages are described below.
n Dead-end filtration is easy to assemble, but the likelihood of clogging is high. This method reduces the risk of cross-flow filtration where the filter membrane is mounted horizontally to the flow direction, but efficiency is also reduced. So-called H-filters (channels structured like an H shape with two inlets and two outlets) take advantage of gravity or the concentration gradient between the sample and a second fluid, but they depend on the diffusion velocity of the particles. Thus, this filtration method is restricted to low flow rates and small sample volumes.
- A more sophisticated method is the use of beads trapped by filter structures or magnetic forces.4,5 These beads can be modified in a number of ways, for example, with biotin or antibodies covalently bound to the bead surface. This leads to high specific filtration results, but requires more effort for the bead handling and higher costs because of the modification.
- An effective method is a dielectrophoresis filter, which attracts particles as a result of their different polarisability. This method allows the separation of dead cells from living cells, but it requires strong inhomogeneous electric fields, which increase the demands on the surrounding equipment and only low flow rates are applicable. No systems are known to have entered the market yet.
- A standard method in the laboratory scale is centrifugation, but only a few compact-disk-like microsystems utlise it. These disk solutions take advantage of the centrifugal force to pump the sample and separate cells, for example, for blood-plasma generation.6,7,8 However, a centrifuge integrated into a static chip system has not yet been achieved. Figure 3 shows a centrifugal microsystem that comprises an electric motor and two chips; one chip represents the fluidic interface and the other the rotator. Combined with automatic positioning and lifting of the rotation chip, this set-up opens up a completely new field for static centrifugal chip systems. The centrifuge design shown in Figure 3 can reach up to 4500 g at 15000 rpm.
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Figure 3: Centrifuge module for sample separation. The rotary chip can spin with 15000 rpm reaching 4500g.
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Cell lysis
Often cell-free parts of the sample are of interest. Therefore the next step is to process the filtrate. However, if the collected cells have to be analysed, they must be lysed to obtain access to their content. One well-known method is chemical lysis using detergents. To avoid interference with downstream process steps these detergents usually have to be removed. This can be easily achieved during DNA purification before polymerase chain reaction analysis or hybridisation assays are performed. In contrast, ELISA assays for protein detection will be severely disturbed.
Thus, for protein analysis, thermal lysis can be performed, which removes the need for detergents. Another alternative is lysis via ultrasound,9 but problems of tight coupling to the chip and heating of the sample have to be resolved. A more sophisticated method is the lysis by electrical fields or electrolysis in which a strong, but short jump of the pH-value is obtained, thus no damage of biomolecules is observed.10
DNA and protein purification
At this point in an assay, the targets are flowing in a preconcentrated manner in a more or less well defined matrix. To allow final detection, purification of the proteins or nucleic acids is usually required to allow reliable and reproducible results. Many well-known methods can be employed at the chip level. Columns filled with specially pretreated beads can be incorporated into the chip to perform nucleic acid purification according to Boom’s method.11 or nucleic acid absorbing bead materials can be used. Problems arising from a bead-based approach include the input and storage of the beads, the need for a homogeneous flow to achieve an equal distribution of the analytes in the bead chamber, and the increase of pressure that may occur because of the packing of the beads (a well-known task even in macroscopic columns for protein or DNA purification). The same approach can be used for the purification of proteins using antibody-coupled beads or other molecules with high affinity to specific proteins.
To circumvent the problems arising from the use of beads, flat polyamide membranes or strips can be integrated onto the bottom of the fluid channel. Sufficient contact time of the analytes with the absorbing material must be ensured. The contact time depends on the volume of the sample as well as the geometry of the channel and the applied flow rate. Because of the characteristic laminar flow pattern in microchannels, low flow rates, small channel depths or structures inducing vortices inside the channel are required.
Outlook
Although only a small number of existing technologies have been mentioned in this article: extraction, filtration, centrifugation and purification, it has shown that the basis for a complete on-chip analysis is available. The real challenge is to bring together the individual modules to build-up easy-to-use and reliable systems with sufficiently high selectivity and sensitivity. Technological questions concerning integration and compatibility will be solved in near future. Thus, expectations that fully integrated lab-on-a-chip systems will enter the market in a short time period are justified.
1. D.R. Reyes et al., “Micro Total Analysis Systems. Introduction, Theory, And Technology,” Anal. Chem., 74, 2623–2636, 2002.
2. D.R. Reyes et al., “Micro Total Analysis Systems. Introduction, Theory, And Technology,” Anal. Chem., 74, 2637–2652, 2002.
3. P. Yager et al., “Microfluidic Diagnostic Technologies For Global Public Health.” Nature, 442, 412–418, 2006.
4. N.V. Zaytseva et al., “Development of a Microfluidic Biosensor Module For Pathogen Detection,” Lab Chip, 5, 8, 805–11, 2005.
5. V.I. Furdui, J.K. Kariuki and D.J. Harrison, “Microfabricated Electrolysis Pump System For Isolating Rare Cells In Blood,” J. Micromech. Microeng., 13, S164–S170, 2003.
6. J. Steigert et al., “Fully Integrated Whole Blood Testing By Real-Time Absorption Measurement On A Centrifugal Platform,” Lab Chip, 6, 8, 1040–4, 2006.
7. S. Haeberle et al., “Centrifugal extraction of plasma from whole blood on a rotating disk,” Lab Chip, 6, 6, 776–81, 2006.
8. M. Madou et al., “Lab On A CD,”Annu. Rev. Biomed. Eng., 8, 601–28, 2006.
9. J.M. Bienvenue et al., “Microchip-Based Cell Lysis and DNA Extraction From Sperm Cells For Application to Forensic Analysis,” J. Forensic Sci., 51,2, 266–73, 2006.
10. D. Di Carlo et al., “On-Chip Cell Lysis By Local Hydroxide Generation,” Lab Chip, 5, 2, 171–8, 2005.
11. R. Boom et al., “Rapid And Simple Method For Purification Of Nucleic Acids,” J. Clin. Microbiol., 28, 3, 495–503, 1990.
Dr Klaus S. Drese is Head of the Fluidics and Simulation Department
Dr Frithjof von Germar is Head of the Fluidics Group
Dr Marion Ritzi is Head of the Bio-Chip Group all at Institut für Mikrotechnik Mainz GmbH, Fluidics & Simulation, Carl-Zeiss-Str. 18-20, D-55129 Mainz, Germany, tel. +49 6131 990 431,
e-mail: germar@imm-mainz.de
www.imm-mainz.de
