Download Microfluidic Tools for Systems Biology

1
Microfluidics Tools for Systems Biology
Victor Castellino

Abstract— The systems approach to molecular biology seeks to
evaluate cell functions by completely characterizing all entities
and inter-relationships within the cellular system. Construction,
perturbation, and analysis of systems biology models requires
automated tools that are capable of high-throughput, parallel
processing.
Further progress, particularly in the area of
proteomics, is greatly hindered by the dimensional and flow
constraints on current technology.
Integrated microfluidic
devices provide a set of tools capable of operating on the scale
demanded by proteomic research, offering increased analytical
precision and requiring pico- to nanoliter reagent volumes. While
several devices have been developed for systems biologists, a lack
of modularity, standard design techniques, and devices for sample
preparation currently prevent microfluidic technology from
becoming commercially viable on a worldwide scale.
Keywords— Microfluidics, Systems Biology,
Preparation, Miniaturized Total Analysis System
development of fundamental principles constraining biological
models, perhaps resulting in greater predictive abilities [1].
Feasibility of the systems biology approach strongly
depends on two factors. The first of these is the availability of
data with which to construct fundamental models. This was
addressed by the largely successful Human Genome Project, a
critical data mining breakthrough. The second factor is the
availability of high throughput analytical tools capable of
spatial and temporal data processing. In this regard, micro
total analysis systems, and microfluidic technology in
particular, promises to provide the fully integrated, automated,
high-throughput tools appropriate to the micro- to-nanoscopic
scale and large breadth of genomic data. [3]
Sample
I. INTRODUCTION
A. Systems Biology
Traditional microbiology has focused on identifying and
cataloguing elements of the genetic machinery behind cell
function. However, recent advances in the development of
high throughput analytical tools, as well as readily available
human genomic data have led to a dramatic shift in biological
thought. The new methodology, termed systems biology,
focuses on examining the relationships between components,
rather than on the nature of the components themselves. [1],
[2].
The systems approach incorporates the results of data
mining and cataloguing efforts into mathematical or other
abstract mechanistic models, thus allowing for a preliminary
analysis of relationships between elements in the system. The
goal of systems biology is to thoroughly characterize system
states by introducing and understanding the effects of
perturbations [3]. These perturbations can include both direct
genetic alterations, such as changes to a DNA base pair
sequence, and changes to environmental variables such as
temperature and concentration. Kitano suggests that analyzing
biological patterns at the systems level will lead to the
Manuscript submitted November 1, 2004. V. Castellino is an M.A.Sc
candidate from the Department of Chemical Engineering and Applied
Chemistry at the University of Toronto, (e-mail: [email protected]).
B. Integrated Microfluidic Devices
In its simplest form, a microfluidic chip is an integrated
device consisting of microchannels and microelectronic
control systems. As the common name “lab on a chip” would
suggest, one goal of microfluidics research is to reap the
potential cost, efficiency, reagent volume and parallel
processing benefits associated with scaling down analytical
processes [4]. Over the past decade, an increasing research
focus on controlled surface interactions, development of
integrated microelectromechanical systems (MEMS) including
actuators and power supplies, gating, valving and sensory
techniques has led to progressively more sophisticated systems
[5], [ 6].
However, the lack of tools and techniques for the
preparation of complex biological or chemical samples for
microfluidic analysis remains an impediment to the
construction of devices appropriate to laboratory use. In
addition, current trends in microfluidic technology have
largely been based on single applications, with industrial
issues such as standardization, modularity, and technology
transfer often ignored. To fully realize the potential of
integrated microfluidic devices, an appropriate methodology
must be developed to ensure that sample preparation,
processing and analysis can all be conducted efficiently with a
standard set of well understood tools. This paper will review
key modular microfluidic technologies and their relevance to
systems biology, as well as discussing efforts towards
component standardization.
2
II. MICROFLUIDICS FOR SYSTEM BIOLOGISTS
A. Genomic Tools
The most recognizable examples of application specific
micro total analysis technology applied to microbiology can be
found in the wide variety of microarrays used in genomic
analysis. Microarray experimentation involves fixing probe
DNA or oligonucleotide strands to a chip surface, then
hybridizing fluorescently labeled sample DNA/RNA to the
probe strands. In this way, intensity of fluorescence at each
fixed point on the chip provides an indication of sample
composition [7]
One of the most important microfluidic tools available to
systems biologists, and in fact to all molecular biologists, is the
polymerase chain reaction (PCR). The PCR technique allows
for exponential amplification of sample DNA through a
cyclical heating and cooling process, where new DNA strands
are formed from free deoxyribonucleotides based on templates
provided by existing strands [5], [7]. Li et al. describe several
approaches to transferring this technique to a portable
microchip. These include partially closed-loop chips with
three temperature-controlled flow zones as shown in figure 1,
and batch reactors. In addition, microfluidic PCR devices
have been incorporated onto chips containing secondary
analytical components, such as capillary electrophoresis [5].
powerful as PCR and gene microarrays used in genomic
research. With limited amounts of sample available covering a
broad spectrum of protein concentrations, analytical precision
and efficient sample use become critical experimental factors
[3], [7]. Estimates for the number of different proteins
produced by humans span an order of magnitude, from a
hundred thousand to a million. Assembling and analyzing
proteomic data as thoroughly as genomic data is beyond the
ability of current analytical tools, being very time consuming
and computationally expensive.
Highly integrated
microfluidic devices that can perform multiple experimental
steps on one chip are well suited to proteomics for several
reasons. First, the scale of the device naturally implies short
diffusion, and hence short analysis times. Second, the scale
also implies minute reagent requirements. Finally, the scale
brings with it a high surface to volume ratio, ideal for reactions
dependent on surface interactions [8]. As an example of such
an integrated device, Gao et al. have constructed a chip that
integrates digestion, separation and identification components
onto a polydimethylsiloxane (PDMS) substrate. It requires
less than a nanogram of sample, with processing time on the
order of minutes [9]
While microfluidic tools for proteomic analysis remain far
behind genomic tools in terms of sophistication, several high
throughput tools for screening and drug discovery have been
developed in the form of bioassays. For instance, Guijt et al.
have reviewed a series of immunoassays based on microfluidic
capillary electrophoresis, including high throughput
multiplexed assays [10]. In addition, Li et al. have reviewed
devices capable of enzyme-linked-immunosorbant-assay
(ELISA), on-chip sensory and control systems,on-chip reactors
for heterogeneous bioassays, and coupling with mass
spectrometry [5].
III. MICROFABRICATION AND MODULARITY
A. Custom Assembly
Figure 1: Schematic of PCR implemented on a chip with
distinct temperature-controlled regions. Adapted from [7].
B. Proteomic Tools
While manufacturing cost and efficiency are certainly
important considerations in the development of any
technology, systems biologists will perhaps be most interested
in the pico-to-nanoliter reagent volumes required for
microfluidic analysis. According to Reyes et al., micro total
analysis systems were originally motivated by a need for
greater analytical precision, rather than for the convenience of
scaling down a laboratory [6].
The scale of microfluidic analysis is particularly appealing
to proteomic researchers. The proteomic field lacks tools as
Even the simplest devices described above were constructed
with a specific application in mind.
The individual
components of each chip are not modular, and can only be
transferred to other microfluidic technologies conceptually. In
effect, every device is custom made. This approach, while
certainly viable in a research and development setting, presents
obvious difficulties to large scale commercial and laboratory
use in terms of standardization and manufacturing precision.
As a result, microfluidic devices have yet to reach the goal of
start to finish experimental automation, as many steps must
still be performed by hand. For example, the immunoassays
reviewed by Guijt et al. still require that prepared sample be
pipetted into input wells on the microchip [10].
3
B. Manufacturing and Design Techniques
In order to clarify the need for greater modularity in
microfluidic research, the basic techniques by which chips are
constructed will be reviewed. Typical substrates include glass,
silicon, and PDMS, with each offering different thermal and
structural properties. Assembly methods have evolved from
microelectronic fabrication techniques, with popular
techniques such as soft lithography often used in laboratory
research [11]. An example of a channel and well cast in epoxy
is shown in figure 2. Extremely precise microfabrication
techniques are required, particularly when microeletromechanical systems (MEMS) and other control elements
are incorporated into the design.
detection mechanisms.
Yadavalli and Pishko present
fluorescence polarization as a successful method for detecting
binding agents in a polydimethylsiloxane (PDMS)
immunoassay chip, as shown in figure 3 [12]. While
constructed as part of an integrated chip, the schematic is
meant to illustrate the detection module in a continuous flow
reactor.
Research into the design of channel geometries for droplet
control, as reported by Tan et al., uses a similar, low-level
approach to microfluidics design. Tan et al. focus on flow
control at a bifurcation junction, and have examined control of
droplet fission, control of daughter droplet concentrations, and
control of droplet circulation [13]. Evaluating droplet flow on
at this level ensures that their results are almost universally
applicable to microfluidic chip design.
Figure 2: Microfluidic channel cast in epoxy (SEM image)
[11].
As suggested above, applying lithographic and casting
techniques to microfluidic device assembly results in a library
of custom made molds, one for each device. The result is that
experimentation with novel techniques, perhaps a new valving
or sensory mechanism, requires prototyping entirely new
designs to replace the old.
The electronics industry has long since focused on modular
design, whether on the scale of individual transistors, or at the
level of integrated circuitry. From an engineering perspective,
this modularity provides the researcher with a toolkit from
which to construct experiments. It further allows for more
precise study of each module as a system with controllable
parameters. Favorable economic implications of modularity
aside, a similar toolkit consisting of reaction, separation,
detection, and valving mechanisms analogous to resistors and
transistors would offer the systems biologist experimental
freedom beyond the current custom design paradigm.
C. Module Development
A low-level research approach, where the basic elements of
the microfluidic system are developed first as building blocks
to more complex systems, has already begun to show
promising results. For instance, the analytical precision with
regards to high-throughput microfluidic technologies stems
from the availability of accurate, integrated sensory and
Figure 3: Schematic of on-chip fluorescence polarization
as part of a microfluidic assay. Adapted from [12].
One area that stands to benefit greatly from the introduction
of modular design is that of chemical engineering unit
operations. Sample preparation techniques involving solvent
extraction, filtration, adsorption, crystallization and sample
concentration are well understood, and have long been used by
chemical engineers for larger scale applications. Grodzinski et
al. point out that current microfluidic technology has focused
on the final steps of the analysis process, requiring purified
sample DNA or protein as an input [11]. According to
Tokeshi et al, the small input volumes and excellent heat
transfer properties of microfluidic chips make chip-based
capillary electrophoresis speeds comparable, if not superior to
the macroscopic equivalent [14]. Adapting traditionally large
scale chemical and biological preparation techniques to the
microscale is therefore an important step in achieving the
desired goal of completely automated, high-throughput parallel
analysis. Approaching the separation techniques as modular
microunit operations (MUOs) simplifies this process. These
are illustrated in figure 4.
4
tools for spatial and temporal analysis with minute input
reagent volumes. While many devices supporting critical
functions such as PCR and bioassays have reached
commercialization, the industry itself is yet to grow beyond its
infancy. A major impediment to further growth is the lack of
standardization, in that microfluidic device assembly currently
requires custom designs from the ground up. A focus on
modularity will benefit both industrial and academic research
interests, allowing for greater coordination between research
groups and reduced manufacturing costs. Most importantly,
however, modular biological sample preparation elements are
required to fully realize the potential of microfluidic devices in
a laboratory setting, eliminating the need for costly and labour
intensive preparation equipment. In time, microfluidics will
ideally come to offer the systems biologist a toolkit as simple
and yet as comprehensive as what an electrical engineer can
purchase at any electronics store.
REFERENCES
Figure 4: Illustration of microfluidic sample preparation
implementations [14]. a) Mixing b) Phase formation c)
Extraction d) Phase Separation e) Filtration f) Heating
Micronics, Inc. of Redmond, WA have begun to develop
such separation systems, capable of handling inputs such as
whole blood and contaminated water. For example, the
Micronics Hematology Cartridge, illustrated in figure 5, is
capable of measuring red blood cell and platelet counts from
whole blood samples. Moreover, Schulte et al. describe the
development of modular, passive mixers, detectors and
separators that do not require external power [15]. This
represents, perhaps, the first step towards point-of-care
applications.
[1]
[2]
[3]
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Figure 5: Micronics Hematology Cartridge [14].
[14]
IV. CONCLUSION
Microfluidic technology promises to revolutionize systems
biology, providing completely automated, high throughput
[15]
H. Kitano, “Looking Beyond the Details: A Rise in System-Oriented
Approaches in Genetics and Molecular Biology,” Curr. Genet., 2002,
vol. 41, pp. 1-10.
T. Ideker, T. Galitski, L. Hood, “A New Approach to Decoding Life:
Systems Biology,” Annu. Rev. Genomics Hum. Genet., 2001, vol. 2,
pp. 343-372.
AD Weston, L. Hood, “Systems Biology, Proteomics, and the Future
of Health Care: Toward Predictive, Preventative, and Personalized
Medicine,” Journal of Proteome Research, 2004, vol. 3, no. 2, pp.
179-196.
A. Manz, N. Graber, H.M Widmer, “Miniaturized Total Chemical
Analysis Systems: A Novel Concept for Chemical Sensing,” Sensors
and Actuators B1, 1990, vol. 1, pp 244-248.
D. Erickson, D. Li, “Integrated Microfluidic Devices,” Analytica
Chimica Acta, 2004, vol. 507, pp. 11-26.
D. Reyes, D. Iossifidis, P.A. Auroux, A. Manz, “Micro Total Analysis
Systems 1: Introduction, Theory and Technology,” Anal. Chem.,
2002, vol. 72, pp 2623 -2636.
G. Sanders, A. Manz, “Chip-Based Microsystems for Genomic and
Proteomic Analysis,” Trends in Analytical Chemistry, vol. 19, no. 6,
pp 364-378.
N. Lion et al., “Microfluidic Systems in Proteomics,” Electrophoresis,
2003, vol. 24, 3533-3562.
J. Gao, J. Xu, L. Locascio, C. Lee, “Integrated Microfluidic System
Enabling Protein Digestion, Peptide Separation, and Protein
Identification,” Anal. Chem., 2001, vol. 72, pp 2648-2655.
R. Guijt, E. Baltussen, G. van Dedem, “Use of Bioaffinity Interactions
in Electrokinetically Controlled Assays on Microfabricated Devices,”
Electrophoresis, 2002, vol. 23, pp. 823-835.
P. Grodzinski et al. “Development of Plastic Microfluidic Devices for
Sample Preparation,” Biomedical Microdevices, 2001, vol. 3, pp 275283.
V.Yadavalli, M. Pishko, “Biosensing in Microfluidic Channels using
Fluorescence Polarization,” Analytica Chimica Acta, 2004, vol. 507,
pp 123-128.
YC Tan, JS Fisher, AI Lee, V. Cristini, AP Lee, “Design of
Microfluidic Channel Geometries for the Control of Droplet Volume,
Chemical Concentration, and Sorting,” Lab on a Chip, 2004, vol. 4,
pp 292-298
M. Tokeshi, Y Kikutani, A. Hibara, K. Sato, H. Hisamoto, T.
Kitamori, “Chemical Processing on Microchips for Analysis,
Synthesis, and Bioassay,” Electrophoresis, 2003, vol. 24, pp 35833594.
T. Schulte, R. Bardell, B. Weigl, “Sample Acquisition and Control:
On-Chip Microfluidic Sample Preparation,” Micronics Inc.,
Redmond, WA.