Microfluidics and microarray based approaches to biological analysis 1

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, oligonucleotide

CHAPTER 1 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

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 1 and 2 provides the separation and that defined by 3 and 4 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 3 and 4 allows for sample material to be pulled across the cross-junction, switching off this voltage and applying one between 1 and 2 pulls material onto the separating channel This allows very small plugs (of pL volumes) of sample to be introduced

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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-to-volume 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

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 microchip-based 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.8However, 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

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

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 do 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

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 transcriptional 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

post-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/SDS-PAGE 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

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,

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.32Furthermore, 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