Chapter CHAPTER INTRODUCTION The interpretation of the human genome requires new tools that can deliver genetic and proteomic information rapidly, in a high-throughput fashion, at low cost and with high accuracy. The sheer repertoire of information within a single cell in terms of genes being expressed and proteins present requires the technology to be ultimately rapid and affordable. These microanalysis devices can usually be classified into two broad categories: microfluidic-based microdevices and microarray-based devices. 1.1 Micro Fluidics-Based Technologies In the past 10 years, microfluidics has progressed rapidly from a simple concept to the basis of new technologies that promise tremendous advantages in the field of biomedical sciences. A general trend in microchip-based separation techniques has been the dominance of electrophoretic over pressure-driven separation techniques. There are probably two main reasons for the bias towards electrophoresis. The application of voltage across the terminal ends of microchannels is much easier to realize from an engineering point of view than the application of a pressure difference, because no moving parts, such as pumps or valves are required. At the same time, depending on the surface properties and the buffer composition, an overall flow of the bulk liquid can be readily induced within the channel network when an electric field is applied. Chapter 1.1.1 Capillary Electrophoresis and Microchip-Based Capillary Electrophoresis 1.1.1.1 Capillary Electrophoresis The feasibility of performing free solution-based electrophoresis in narrow tubes was first demonstrated by Hjerten in 1967.1 However, the real breakthrough came from the work of Jorgensen and Luckas, where, using small capillaries and high electric fields, they demonstrated the feasibility of high-speed, high-resolution separations in glass capillaries.2 1.1.1.2 Microchip-Based Capillary Electrophoresis In 1992, Harisson and Manz showed that small bore capillary channels, with inner dimensions of 30 × 10 µm, etched in planar glass substrates, could be used to perform on-chip capillary electrophoresis, also termed as micro capillary electrophoresis (µCE).3 Figure 1.1 shows a basic chip-based device for electrophoretic separations. The channel defined by points and provides the separation and that defined by and is the injection channel. The ends of the channels contain reservoirs for waste, buffer or sample. These also provide access for the electrodes. The channels may be filled with a buffer of constant pH or with sieving material such as polyacrylamide gel. Applying a voltage between point and allows for sample material to be pulled across the cross-junction, switching off this voltage and applying one between and pulls material onto the separating channel. This allows very small plugs (of pL volumes) of sample to be introduced. Chapter Sample reservoir Injection Cross Buffer reservoir Buffer waste Separation Channel Sample waste Figure 1.1. Schematic drawing of a microchip based electrophoretic device The advantages conferred by such microfluidic-based systems are numerous and wide ranging. The miniaturization leads to less reagent consumption, and ultimately the fabrication of such systems will be economically advantageous compared to traditional analytical systems. Other advantages arise as a result of the higher surface area-tovolume ratio of the systems, giving dramatically increased performances: improved thermal diffusion resulting in fast cooling and heating of fluidic elements. This also means that, for example, in electrophoretic separations, higher voltage gradients may be used without Joule heating of the system as the power is more efficiently dissipated within the microstructure. Micro scale-based separations thus offer improved speed and efficiency compared to conventional electrophoretic-based separations. The channel dimensions and flow rates typically employed in microfluidic systems generally lead to laminar flow. As a result, band broadening and increased pressure from turbulence are avoided. Faster separation, achieved by miniaturization, further leads to less diffusional band broadening. The efficiency of electrophoretic and chromatographic separation, measured in the number of theoretical plates, is proportional to the length of the separation channel over the diameter of channel. This Chapter means that reduction in size can be successfully facilitated without a loss in the number of theoretical plates. 1.1.2 Microchip-Based Analysis Applications The main field of application for microchip-based separations is the analysis of biologically relevant molecules, namely DNA, oligonucleotides, proteins and peptides, with the separation of nucleic acids being one of the leading applications of microchipbased analysis. One of the driving forces behind this development of microchip-based DNA analysis was the Human Genome Project and the many follow-up projects it spawned with the emphasis on efforts for high-speed sequencing. Although the technique currently used in most commercially available DNA sequencers- Capillary Electrophoresis- is much faster than slab-gel electrophoresis, micro CE based sequencing can hasten the process considerably.4 DNA sequencing, one of the most challenging tasks in DNA separation due to the very high resolving power needed, has been developed in a high throughput format using a microchip device containing 96 channels.5,6 Polymers such as polyacrylamide, used in slab gel electrophoresis can efficiently be transferred to the microchip format in which case capillaries need to be derivatized to remove the electroosmotic flow to allow for efficient size based separation of nucleic acids. Matrix-free DNA analysis has also been reported and a nanofluidic channel was designed and fabricated to separate long DNA molecules based on the so-called “entropic traps” principle.7 High speed protein separation has also been developed on microchip based devices.8 However, most of the current technologies used to separate proteins still rely on 2D gel electrophoresis and these are not as easily transferable to microchip format as slab gel Chapter DNA separation. Some challenges still need to be overcome, and as a result there is still a widespread interest in developing 2D microchip based protein separation, as this would dramatically shorten the separation time.9,10 1.1.3 Developing a Fully Integrated Lab-on-a-Chip Device One of the aims, when designing a complex microsystem, is to develop complete systems allowing various stages of DNA analysis to be performed on a single microdevice. These stages include, for instance, PCR amplification, DNA preconcentration, restriction digest, hybridization, and may include more complex “building blocks” such as microvalves, microreactors as well as various detection methods. One of the major expectations for microchip separation devices is that they will dramatically increase the sample throughput, both by reducing the time per analysis and by processing several analyses in parallel; the goal being to achieve a higher degree of complexity by integrating complex elements such as valves, mixers in order to realize what is commonly called a “lab-on-a-chip”. Various levels of integration have so far been reported and a wide range of analytical reactions such as nucleic acid separation by capillary electrophoresis (CE), DNA sequencing, polymerase chain reaction amplification, immunoassays, or single nucleotide polymorphism (SNP) analysis have already been performed on a microscale format. However, in most cases, the complete integration of these various techniques together with the separation step onto a single chip is not taken into account and often one or several of the steps are still performed off-chip. Chapter 1.1.4 Limitations, Issues to Be Addressed Progress on the construction of fully integrated chemical systems has lagged behind compared to the development of single components since the integration of these “building blocks” remain challenging. Currently, sample preparation is often the most difficult step in an assay, and is therefore typically performed separately from the reaction and detection steps, with so far very few reports of on-chip sample preparation.11 1.2 Array-Based Technology 1.2.1 DNA Microarrays for High Throughput Genomics Studies New technologies have been developed for rapid sequencing of DNA, and with the recent completion of the Human Genome Project,12,13 tools are needed to help in the understanding of the functions of these sequenced genes. Unfortunately, the billions of bases of DNA sequences not tell us what all the genes do, how cells work, and how cells form organisms. The goal is not simply to provide a catalogue of all the genes and information about their function, but to understand how the components work together to direct cells and organisms. Among the most powerful and versatile tools currently available for genomics are DNA microarrays. DNA microarrays consist of large numbers of DNA molecules spotted in a systematic order on a solid substrate and finds its roots in the form of southern blot.14 DNA microarrays work by hybridization of labeled RNA or DNA in solution to DNA molecules attached at specific locations on a surface. They are commonly used either to monitor expression of the arrayed genes in mRNA populations from living cells15,16 or to detect DNA sequence polymorphisms or mutations in genomic DNA.17 Chapter DNA microarrays are usually distinguished by the size of arrayed DNA fragments, the methods of arraying, the chemistry and linkers for attaching DNA to the chip. Two DNA chip formats are currently widely used, these are the cDNA array format18 and the in situ synthesized oligonucleotides array format.19 The probes are a reverse complement of target regions on mRNA (or cDNA) whose concentration or expression level is monitored through hybridization. In the first case, the probes are obtained as PCR products of intact cDNA (300 – 1000 base long) spotted onto the slide surface. In the second case the short oligonucleotides (20 – 30 base long) are synthesized in situ. While making arrays with more than several hundred elements was until recently a significant technical achievement, arrays with more than 250,000 probes20 or 10,000 different cDNAs21 per square centimeter can now be produced in significant numbers. Alternatively, long oligomers (50 – 70 bp) have also recently been used for DNA microarrays.22 Long oligomers show the same sensitivity as cDNA PCR products in the detection of the target genes. 1.2.2 From Genomics to Proteomics 1.2.2.1 Limits of DNA Microarray-Based Strategies DNA microarray-based strategies allow for a detailed understanding of the regulation of biological systems. However, such methods provide no information about posttranscriptional control of gene expression, changes in protein expression levels, changes in protein synthesis and degradation rates or protein post-translational modifications. In addition, recent studies suggest that mRNA levels correlate poorly with protein expression levels.23 Hence, the current research shifts from genomics to proteomics. Proteomics includes not only the identification and quantification of Chapter proteins; but also the determination of their localization, modifications, interactions, activities and ultimately, their function.24 Proteins, however, are much more complex than nucleic acids. Unlike DNA, proteins get phosphorylated, glycosylated, acetylated, etc. A single gene can encode multiple different proteins; these can be produced by alternative splicing of the mRNA transcript by varying translation start or stop sites, or by frameshifting during which a different set of triplet codons in the mRNA is translated. All of these possibilities result in a proteome estimated to be an order of magnitude more complex than the genome. Although it was concluded from the Human Genome Project that there are about 30,000 – 40,000 genes in human, it has been estimated that the human proteome could contain from as few as 100,000 proteins to as many as a few millions. In addition, proteins respond to altered conditions by changing their location within the cell, getting cleaved into pieces, and adjusting their configuration as well as changing the molecules they bind to. 1.2.2.2 Current Strategies for High Throughput Proteomics The most widely available tool for proteome analysis, 2D gel electrophoresis (2DE) has been available for more than 25 years.25 To date, most proteomics experiments have relied on two-dimensional gel electrophoresis using isoelectric focusing/SDSPAGE and mass spectrometry for their separation and detection methods respectively.26 Unfortunately, despite the considerable resolving power of 2DE, this technology has so far fallen far short of the ultimate goal of displaying in one experiment an entire cell or tissue proteome. Several classes of proteins have proven especially resistant to analysis by 2DE, including low and high molecular mass proteins, membrane proteins, proteins with extreme isoelectric points and low abundance proteins.27 Indeed, with the capacity and sensitivity of 2DE having been Chapter pushed to their limits, alternative and/or complementary separation strategies must be developed in order to permit the characterization of the proteome. Although proteins are actively involved in various biological activities, they must interact with other molecules to fulfill their roles. Thus, the identification of binding partners is crucial to understanding the function of a protein. The two-hybrid assay has proven to be one of the most efficient techniques for finding new interactions.28 The procedure is simple, inexpensive and has the important advantage of being unbiased (i.e. no previous knowledge about the interacting proteins is necessary for a screen to be performed). However, the system also has a reputation for producing a significant number of false positives that require cumbersome analysis to separate the “wheat” of true interactions from the “chaff” of false positives. 1.2.3 Protein Microarrays for High Throughput Proteomics Proteins have complex three-dimensional conformations that have direct impact on their function and binding properties and they usually function in complexes with other proteins or embedded in membranes. Proteins interact with other molecules- other proteins, nucleic acids, and small ligands – and the physico-chemical nature of these interactions is much more diverse than that of nucleic acid hybridization. Because of all these complexities, new non-conventional approaches to study protein interactions in a microarray format are currently being explored. An early application of the array format for proteomics was the parallel synthesis of peptides using a 96-microtiter plate format originally described by Geysen et al.29 SPOT synthesis uses a similar chemistry, but takes advantage of the abundant hydroxyl moieties present on cellulose filter paper. This method has proved versatile and has been successfully used to investigate protein interactions with other proteins, Chapter DNA, as well as kinase activity. The low density of arrayed substrate is however a drawback for its development and the number of peptides bound to the surface was later greatly enhanced by combining solid phase synthesis with photolithographic techniques and an array of 1024 peptides was synthesized in 10 steps.19 Even though this allows for arrays of very high density to be developed, this strategy remains very expensive and rather inflexible. Following the wide success of DNA microarrays, there has been a wide interest in trying to extend the technologies developed in the mid 90s to fabricate protein, peptide, and small molecule arrays for high throughput proteomics. Most of the surfaces used to generate microarrays are made from glass, although plastics, gel pads, silicon and polymer membranes have also been used. Depending upon the different formats adopted for fabrication, the chips may be classified into three categories: slides, porous gel pads and microwells - microstamps, with glass slides being the surface of choice because of its known chemistry and easy functionalization. A number of chemistries have been developed to array these proteins; small molecules and peptides ranging from simple non-covalent surface interactions with hydrophobic or positively charged (poly-Lysine, aminosilane) surfaces30 to site-specific immobilization.31 Sophisticated chemistry has also been developed by companies and research groups to meet the specific needs for immobilizing and stabilizing proteins on microarrays.32 Furthermore, hydrogel modifications33 can be used to prevent the immobilized proteins from drying out. For detection, the same CCD-based fluorescence detection used for DNA microarrays is currently used for protein arrays. Recently, Surface Plasmon Resonance (SPR) has been reported.34 This detection method presents the additional advantage of being able to detect and quantify binding events by using changes in the refractive index of the surface that are caused by increases in mass. There is currently 10 Chapter no strategy available for amplification of proteins similar to PCR amplification for DNA and the amount of proteins obtained might not be sufficient for efficient detection. The rolling circle DNA amplification strategy (RCA) developed for ultrasensitive fluorescence based antigen detection is a promising high-end detection technology35 and it was recently applied to protein microarrays.36 Protein microarrays are generally classified in two broad categories. The first category, called protein-profiling arrays, usually consists of antibody arrays in which antibodies prepared against different proteins or epitopes are spotted onto slides. By incubating these arrays with protein mixture, one can rapidly profile the presence of proteins of interest in a way similar to DNA microarrays. The second category, called protein function arrays, consists of non-antibody protein microarrays in which sets of proteins, enzyme substrates or small molecules are spotted at high density onto a slide. By incubating these arrays with proteins, small molecules, probes, one can screen for protein-protein, protein-small molecule interactions, as well as enzymatic activities. 1.2.3.1 Protein Profiling Arrays The measurement of individual protein expression levels has traditionally been carried out using two dimensional gel electrophoresis. These offer ease of use and adequate sensitivity but they lack scalability. Microarrays of immobilized antibodies for multiplex immunoassay can alleviate these drawbacks and microarray-based ELISA have been reported.37,38,39 The dual labeling previously used for DNA microarrays was used for the parallel detection and quantitation of proteins to measure the concentration ratio of each protein in the two samples by labeling the two samples with two different dyes.31 A similar approach was used for the profiling of cancer cells: 146 different antibodies were arrayed and incubated with fluorescently labeled cell lysates.40 An 11 Chapter autoantigen microarray was recently reported by Robinson et al to perform large-scale multiplex characterization of autoantibody responses directed against structurally diverse autoantigens.41 Arrays were incubated with patient serum labeled with a fluorescent dye. An allergen microarray containing 94 purified allergen molecules has also recently been developed.42 Antibodies are the most prominent capture molecules used to identify targets. However, owing to the labor-intensive nature of monoclonal antibody production, the development of other alternatives has become crucial. One very promising approach in this field is the phage display technique, combined with highly diverse fully synthetic libraries, to generate artificial antibodies.43 Another strategy is the generation of highly specific oligonucleotides.44,45 Such oligonucleotides derived from an in vitro evolution process called SELEX (systematic evolution of ligands by exponential enrichment) are referred to as “aptamers” and appear promising as new array probes.46 Another approach for protein profiling consists in immobilizing the cell extracts on the slide and probing these with labeled antibodies.47,48 Finally, by combining protein array with MS, the ProteinChip technology has also been used for protein profiling.49 The diffusion limit in reaction kinetics remains a major issue in the development of protein profiling arrays. To overcome this problem, Xu et al. developed a filtrationbased protein microarray.50 Proteins were printed onto protein-permeable nitrocellulose filter membranes, which were placed in a customized filtration apparatus for flow-through assays. This strategy improved the overall reaction kinetic rate by 10fold. Toegl et al. used microagitation to shorten incubation times.51 Nanopumps integrated in a cover slip substitute produced surface acoustic waves, which caused mixing of the solution. Applied in protein microarray analyses, this system resulted in shorter incubation times and much higher signal intensities. 12 Chapter 1.2.3.2 Protein Function Arrays Proteins are actively involved in various biological activities, and must interact with other molecules to fulfill their roles. Thus, the identification of protein binding partners is crucial for the understanding of protein functions. Hence, another class of arrays, called protein function array, has been developed to study interaction of proteins with small molecule ligands, peptides, DNA, or other proteins. In parallel, arrays have been developed to study the enzymatic activities of enzymes, especially kinases. In a proofof-concept experiment, McBeath et al arrayed proteins onto functionalized glass slides and probed them with fluorescently labeled proteins and small molecules to screen for protein-protein and protein-small molecule interaction.52 The microarray format allows for the high throughput screening of protein interaction with proteins, and ligands, and presents the additional advantage of requiring only very small quantities of the sample.53,54 The same group later used this strategy in a very high throughput fashion. By arraying more than 3000 small molecules arrayed onto slides, they were able to screen in a very high throughput fashion for potential protein-small molecule interaction and were able to identify one of these small molecules as being inhibitor of the yeast protein Ure2p.55 Membrane proteins are notoriously a lot more difficult to work with since they are stable and retain their biological activity only in a membranelike environment. In addition, they are typically insoluble under physiological conditions and denature when arrayed onto a glass surface. However, by arraying membrane proteins onto amine slides covered with a layer of lipids, Fang et al showed that proteins retain their activity and membrane protein arrays can be generated.56 In addition to protein arrays, carbohydrate arrays have also been recently reported.57,58 In the first case, 50 different glycans were immobilized onto nitrocellulose coated surfaces, whereas in the second case, carbohydrates were synthesized with a 13 Chapter cyclopentadiene moiety for site-specific binding onto hydroquinone functionalized slides. In parallel, microarrays have also been used to study enzyme activity. By arraying kinase substrate onto slides53 or into microwells,59 one can potentially screen in a high throughput fashion for enzyme activity, and 119 out of the 122 yeast kinases were studied.59 Finally, one can eventually screen for protein, small molecule interaction with the whole proteome of any organism of interest. This was demonstrated, in a “tour de force” experiment, by Zhu et al.31 They expressed 5,800 yeast proteins with a GST tag for purification purposes and with a His tag for sitespecific immobilization onto Ni-NTA functionalized slide. This allowed for very high throughput screening of protein-protein, protein-lipid, and protein-nucleic acid interactions. Instead of arraying the proteins or ligands, DNA microarrays have also been used for proteomics studies, for instance to study DNA binding proteins.60 DNA microarrays combined with chromatin immunoprecipitation have also been used to identify transcription factors.61 Addressable small molecule microarray can also be obtained from DNA microarrays by hybridizing them with libraries of small molecules tethered to peptidonucleic acids (PNA) tags62 and with mRNA-protein fusions. 1.2.3.3 Non-Conventional Protein Arrays Most of the small molecules, peptide, and protein arrays developed to date are based on the strategies developed for DNA microarrays, but since proteins are much more difficult to handle than DNA, non-conventional strategies have also been developed. When fabricating protein microarrays, the stability of the spotted proteins is a main concern since these tend to denature and lose their activity. Sabatini et al demonstrated how protein arrays could be generated in a matter of hours from extremely stable DNA 14 Chapter microarrays.63 Full-length open reading frames of the gene in expression vectors are printed at high density on a glass slide along with a lipid transfection reagent. The slide is placed in a cell culture plate and the microarray of cDNAs is covered with a lawn of adherent cells. Cells growing on top of the DNA spots are reverse transfected, driving expression of specific proteins in spatially distinct groups of cells. The phenotypic effects of this “reverse-transfection” of hundreds or thousands of genes can be detected using cell-based bioassays. Applications were demonstrated for identification of drugtarget interactions and for evaluation of phenotype changes resulting from the expression of specific proteins in the cells. However, as attractive as this approach may seem, it still has its share of drawbacks, and one of the current limit being that only surface proteins could be studied. Tissue microarrays (TMAs) are miniaturized collections of arrayed tissue spots on a microscope glass slide that provide a template for highly parallel localization of molecular targets, either at the DNA, RNA or protein level.64 Construction of TMAs is achieved by acquiring cylindrical core specimens from up to 1000 fixed and paraffin-embedded tissue specimens and arraying them at high density into a recipient TMA block.65 These arrays provide high throughput in situ analysis of specific molecular targets in hundreds or thousands of tissue specimens at once. Using dip-pen nanolitography, protein nanoarrays with 100 – 350 nm features were also fabricated.66 These nanoarrays exhibit almost no detectable nonspecific protein binding and can be screened easily by atomic force microscopy. 1.2.3.4 Limits of Current Array-Based Proteomics Approaches Much of the development of protein arrays has been done by analogy with DNA microarrays. However, proteins are very different, and this resulted in some intrinsic problems for the strategies developed so far to fabricate protein arrays. One issue is the 15 Chapter stability of the spotted arrays, DNA is very stable and once spotted, arrays can be stored for long period of times. However, proteins and antibodies denature and lose their biological activity and the lifetime of protein chips once “spotted” still remains to be determined. Both protein profiling arrays and protein function arrays have also their intrinsic current limitations. The ideal protein-profiling array would be a large array of high affinity, high specificity protein ligands, one for each protein in the proteome of interest. This is, however, very challenging due to the very long time needed to generate antibodies. In reality, the task is even more challenging since the detection of different post-translationally modified forms of a protein is one of the principal advantages of moving from nucleic acid to protein-based arrays. A major challenge remains the rapid and efficient isolation of high affinity and specificity protein ligands. In addition, on the contrary to DNA proteins tend to associate with one another. This leads to a complication in the design of ligand discovery strategies. Protein-function arrays also have their share of limits. There are currently two main problems limiting the development of high throughput protein function array: established methods for DNA amplification are available, but none exist for small molecules, peptides and proteins. The limiting step in creating protein arrays, especially those which aim to be global, is the production of the huge diversity of proteins which will form the array elements. In addition functional molecules based on small molecules, peptides and proteins not attach to chips easily and new strategies have to be developed for sitespecific immobilization of proteins in order to ensure they retain their biological activity. 16 Chapter 1.3 References Hjerten, S. Chromatogr. Rev. 1967, 9, 122 Jorgensen, J. W.; Luckas, K. D. Anal. Chem. 1981, 53, 1298 Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Ludi, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253 Woolley, A. T.; Mathies, R. A. Anal. Chem. 1995, 67, 3676 Mathies, R. A.; Huang, X. C. 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Chem. 19 81, 53, 12 98 3. Horton, H.; Brown, E. L. Nat. Biotechnol. 19 96, 14 , 16 75 16 DeRisi, J. L.; Iyer, V. R.; Brown, P. O. Science, 19 97, 278, 680 17 Hacia, J. Nat Genet. 19 99, 21, 42 Chapter 1 18 18 . usually be classified into two broad categories: microfluidic -based microdevices and microarray- based devices. 1. 1 Micro Fluidics -Based Technologies In the past 10 years, microfluidics has progressed