Microfluidics and microarray based approaches to biological analysis 1

Chapter 1
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.





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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.

2


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Separation Channel
1
3
2
4
Sample
reservoir
Buffer
reservoir
Sample
waste
Buffer
waste
Injection Cross
Separation Channel
1
3
2
4
Sample
reservoir

Buffer
reservoir
Sample
waste
Buffer
waste
Injection Cross
Separation Channel
1
3
2
4
Sample
reservoir
Buffer
reservoir
Sample
waste
Buffer
waste
Injection Cross
Separation Channel
1
3
2
4
Sample
reservoir
Buffer
reservoir

Sample
waste
Buffer
waste
Injection Cross





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

3

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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.
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

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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.




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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 cells
15,16
or to detect DNA sequence
polymorphisms or mutations in genomic DNA.
17


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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 format
18
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 probes
20
or 10,000
different cDNAs
21
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 post-
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

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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

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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,

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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) surfaces
30
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 modifications
33
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

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Chapter 1
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
technology
35
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

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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 filtration-
based 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 10-
fold. 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.

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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 proof-
of-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 membrane-
like 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

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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 slides
53
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 site-
specific 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) tags
62
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

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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 drug-

target 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

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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 do not attach to chips easily and new strategies have to be developed for site-
specific immobilization of proteins in order to ensure they retain their biological
activity.




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17
1.3 References


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