Lab-on-a-Chip. Miniaturized Systems for (Bio) Chemical Analysis and Synthesis

380 Pages·2003·15.02 MB·English

by  R. Edwin Oosterbroek and Albert van den Berg (Eds.)

Preface
A little more than 10 years ago, the concept of Micro Total Analysis
Systems (JLITAS) was proposed for the first time by Andreas Manz. Together
with the other 3 pioneers of the [iTAS field (Harrison, Ramsey and Matthies) the
"fab four" clearly demonstrated the advantages of using micro fabricated
devices in particular for capillary electrophoresis applications on chip. Since
then, the area of |iTAS has expanded rapidly, as illustrated by the increase of
attendance of the [iTAS conferences (from some 150 in 1994 to over 800 in
2002). It has appeared that the ^iTAS concept is applicable on a much broader
scale than the initial functional integration of different analysis components on a
chip. Downscaling of individual ftmctions like reactors, separation columns,
fluid-handling devices etc. could change the world of conventional chemistry to
studies done in a "Lab-on-a-Chip". In particular the possibility to handle fluids
(liquids) on the nanoliter and even picoliter scale ("microfluidics") has widened
the scope of JLITAS. As a consequence, "Lab-on-a-Chip" better covers the
research and emphasizes the strong impact of miniaturization and integration on
fluidics and chemical engineering.
In spite of these rapid new developments, no textbooks have been available
until now that provide an overview over the current status of Labs-on-a-Chip
and enable newcomers in the field to get a quick entry. The proceedings of the
jLiTAS meetings, though well appreciated, only present a collection of brief and
temporary reports. For this reason we have approached several leaders in the
field with the request to write down in more depth an overview of their recent
developments. We have chosen to provide a wide overview over the different
aspects of various areas of the Lab-on-a-Chip developments, instead of
highlighting one specific aspect. The result of this is a nice and comprehensive
picture of four major elements of Lab-on-a-Chip systems, viz. technologies,
methods, cell/bead-based systems and applications.
The technology section starts off with two contributions focusing on
polymers (hydrogels and plastics), currenfly being very popular among Lab-on-
a-Chip researchers and developers. The third contribution gives an overview of
the "traditional" substrate materials, silicon and glass. The next chapter
exemplifies the important role of surface chemistry in microfluidics, whereas the
last contribution gives a nice example of how plastic technology is used to go
from principle to production.
In the second section, three major methods are described: transverse
diffusion, a phenomenon offering many opportunities and of major importance
in laminar flow driven microsystems, is discussed in an important first chapter.
The second contribution deals with methods for nano- and picoliter handling,
another exclusive feature of miniaturized, integrated systems, whereas the third
contribution demonstrates how a classical technique (flow injection analysis)
can be transformed for use in a Lab-on-a-Chip.
The third section is dedicated to cell and bead applications and handling in
microsystems. In the first chapter, bead handling for biotech applications is
discussed, using microfabricated elements. The second and third contribution
both relate to the promising and fast growing research in on-chip cell sorting and
counting, albeit using different principles.
Finally, in the last section, a variety of Lab-on-a-Chip applications is
presented. After an impressive demonstration of the enormous power of
capillary electrophoresis on chip coupled to parallellization in the first chapter,
the important application area of microfluidics coupled to mass spectrometry
(MS) is illustrated in two thorough contributions, focusing on protein analysis
and MALDI interfacing respectively. The next two contributions focus on the
topic of chemistry on chip, a topic that clearly demonstrates the changes from
conventional chemistry towards miniature Labs-on-Chip. It is very interesting to
see how the microfluidic format also proves to be very promising for micro-
synthesis apart from the analysis. The last two applications are in the rapidly
growing areas of medical diagnostics and drug delivery. Like many of the
previous applications, is not a very daring prediction that these application areas
may well prove to be close to commercial successes.
It is our hope and expectation that readers enjoy the book, and get an overview
of the possibilities and developments of Labs-on-a-Chip. With this book,
newcomers in the field can make a quick start to become familiar with the large
variety of technologies, methods and applications. We wish you a very inspiring
use of the first Lab-on-a-Chip book!
Edwin Oosterbroek
&
Albert van den Berg
(editors)
Lab-on-a-Chip
R.E. Oosterbroek and A. van den Berg (eds.)
© 2003 Elsevier B.V. All rights reserved.
Hydrogels and polymers as components of a lab on a chip
J. M. Bauer"'' and D. J. Beebe*"
^Theoretical and Applied Mechanics Department, The University of Illinois at
Urbana-Champaign, Urbana, IL 61801
^Biomedical Engineering Department, University of Wisconsin-Madison,
Madison, WI 53706
1. INTRODUCTION
As an addition or alternative to silicon based microelectromechanical
systems (MEMS) fabrication, hydrogels and polymers can prove useful in micro
scale Lab-on-a-Chip applications. This usefulness stems from the fact that
hydrogels and polymers range from mechanically soft to hard, have varying
degrees of porosity, and are able to be formed from an initially liquid mixture. In
addition, hydrogels can act as a sensor or actuator. Consequently, a variety of
system components, such as the walls of a microfluidic network, valves to direct
flow through the network, or sensors to signal the occurrence of a specific event,
can be created.
Hydrogels are three-dimensional networks of polymers that are capable of
retaining solvents. A subset of hydrogels, stimuli-responsive hydrogels, have the
ability to discontinuously change size at a certain level of stimulus (Figure 1).
This property allows stimuli-responsive hydrogels to function as actuators, since
the size change can be hamessed to perform useful work. A single hydrogel
structure can function as a complete feedback system, simultaneously sensing
and reacting to stimulus levels. These properties have attracted a number of
research groups to investigate the applications of hydrogels in microdevices
[1-4].
In general, the time and force response of stimuli-responsive hydrogels is
controlled by the rate at which the stimulus is able to penetrate the hydrogel
network and the rate at which the network is able to change its volume in
response to the stimulus. A combination of chemical and mechanical parameters
determines the resulting rate of volume change. Examples of chemical
parameters are the characteristics of the solvent, the specific polymers that
constitute the hydrogel network, and the conditions used to form the polymer
network. For a given chemical configuration, mechanical parameters such
as the size and shape of the hydrogel and physical constraints on the hydrogel
will affect the response.
This chapter will focus on the engineering aspects of incorporating stimuli-
responsive (pH-sensitive) hydrogels into microscale devices. pH-sensitive
hydrogels discontinuously change size at certain levels of pH. Hydrogels that
have been developed to be responsive to temperature, light, electric fields,
antigens, and other stimuli should be able to be incorporated into microfluidic
devices in a similar manner [5-8]. Thus, confining the discussion to pH-
sensitive hydrogels does not alter the applicability of the methods and concepts
presented here.
CD
E
3
Stimulus collapsed swollen
a b
Fig. 1. Schematics of the behavior of stimuli-responsive hydrogels. (a) Graph of the
volume of a stimuli-responsive hydrogel as the stimulus is varied. Certain types of
stimuli-responsive hydrogels swell (solid line), while others contract (dotted line),
as the amount of stimulus increases beyond a threshold value, (b) Illustration of the
polymer network in a contracted and expanded state.
2. FABRICATION OF MICROSYSTEM COMPONENTS
2.1. Introduction
The formation of hydrogel and polymer components through light activated
polymerization was selected as the means of incorporating hydrogels into
microsystems because it provides several key advantages. First, the size and
shape of the resulting hydrogel component is controlled by lithography; only the
liquid monomer solution exposed to light solidifies into a polymer network.
Second, lithographic techniques are used in the construction of MEMS devices,
facilitating integration with existing processes.
To create a system from hydrogel components formed in this manner, a
universal construction platform with a wide shallow cavity and fixed external
connection points and dimensions is utilized. If a portion of the cavity is
transparent to light, hydrogels can be polymerized inside the cavity to perform
various functions. This approach allows the plat-form and external connection
points to remain constant, as new systems can be created by polymerizing
hydrogel components in different configurations inside the cavity.
2.2. Liquid phase photopolymerization
Directing the light required for polymerization of a liquid monomer
mixture with a photo mask creates structures inside transparent micro devices
(Figure 2) [9]. A cavity is first filled with the liquid monomer mixture and a
photo initiator, and then exposed to light of the proper wavelength to initiate
polymerization. The polymerization time is a fiinction of the monomers, photo
initiator, optical characteristics of the micro device cavity, and light intensity.
For the polymerizable mixtures used, intensities in the tens of milliwatts per
square centimeter have produced polymerization times in the tens of seconds for
cavities that are approximately 200 microns deep. After the polymerization is
complete, the cavity is flushed with an appropriate solvent, typically water or
methanol, to remove the un-polymerized solution.
a be
Fig. 2. The light initiated polymerization of a Uquid monomer mixture can be controlled
by the transparent patterns in an otherwise opaque photo mask, a) Schematic of the
polymerization procedure, b) A single polymerized structure with both curved and
straight-line segments. Scale bar = 250 |im. c) Multiple structures that were
polymerized simultaneously. Scale bar = 500 ^m. The photo masks used to
polymerize the structures are shown at a smaller scale in the upper right hand
portion of the image {D.J. Beebe et al. Nature 404, 2000 - reproduced with
permission [9]).
This procedure produces structures with sidewalls at various angles with
respect to a perpendicular drawn from the photo mask surface [10,11]. A variety
of experimental setups and polymerization conditions have produced sidewalls
from near vertical to at most a 10% difference between the size of the top and
bottom of the structure, with the top larger than the bottom. The sidewall profile
is a function of both optical and chemical effects. Optical effects include the
thickness of the transparent device top, the degree to which the light is
coUimated, and reflections fi-om material interfaces. Chemical effects involve
diffusion during polymerization and the difference in optical properties between
the liquid monomer and the polymerized hydrogel. Initial studies suggest that
non-collimated light is the most significant factor governing the sidewall profile;
however, the complete parameter space of chemical and optical effects has not
yet been fully analyzed.
Both pH-sensitive hydrogels and polymers have been polymerized in
microdevices with the above procedure. The polymers are rigid and serve as
nonfunctional components, for example, as the sidewalls of channels. One pH-
sensitive hydrogel that we have used is formed from a mixture of acrylic acid
(AA) and 2-hydroxyethyl methacrylate (HEMA) (in a 1:4 molar ratio), ethylene
glycol dimethacrylate (EGDMA) (1 wt %) and a photo-initiator (3 wt %,
Irgacurer 651).
mi
Solid Object
c d
Fig. 3. The concept of surface tension driven foraiation of curved microstnictures and
images of polymerized stnictures. a) A solid object in contact with a liquid
interface forms smooth, curved menisci. The middle solid object is hydrophobic,
while the others are hydrophilic. b) A "wall" created with a 50 ^m thick sheet of
transparency plastic, c) A "volcano" with a cone-shaped interior formed using a
drawn glass capillary, d) A "column" created with tubing.
This mixture, after polymerization, produces a hydrogel that swells in basic
solution and contracts in acidic solution. The structural material normally
consists of a mixture of isobomyl acrylate (IBA), 2,2-bis[p-2'-hydroxy-3'-
methacryloxypropoxy) phenylenejpropane or tetraethyleneglycol dimethacrylate
(TeEGDMA), and Irgacure® 651 as the photoinitiator. These monomers
produce rigid structures with fast polymerization kinetics; typical
polymerization times are less than one minute.
2.3. Surface tension driven fabrication
In contrast to the structures in section 2.2 with sidewalls defined by optical
properties during polymerization, polymerized hydrogels and polymers can also
have curved sidewalls [12]. If a photopolymerizable liquid meniscus is
polymerized, the resulting shape will also be curved, provided the
polymerization process does not significantly alter the meniscus shape (Figure
3a). A liquid meniscus profile is a function of the surface tension of the
meniscus, the density difference across the meniscus, and the contact angle
between the meniscus and the solid object creating the meniscus. All other
parameters being equal, a taller meniscus is formed with a smaller contact angle,
higher surface tension, or a lower density difference.
While the parameters affecting the shape of a liquid meniscus are known,
the changes in surface tension, contact angle, and density that occur during
polymerization are not as well understood. Consequently, it is difficult to
determine the exact result of polymerization on a given photopolymerizable
liquid. However, two photopolymerizable liquids, the structural polymer of
secfion 2.2 and a commercially available optical adhesive (Norland Optical
Adhesive), polymerize to form curved, three-dimensional solid structures
(Figure 3b - 3d). One potential application of structures created in this
manner is in the area of transdermal drug delivery. An array of curved needles
could be fabricated in minutes at a lower cost than traditional techniques, such
as electroplating. Although a given photopolymerizable liquid will need to be
tested to determine a relationship between the parameters influencing the
polymerized menisci, this technique should be able to be applied to a number of
other photopolymerizable liquids. Polymerizing stimuli-responsive hydrogels
with curved features in place of the inert polymers depicted here would produce
unique fiincrionalities. For example, a stimuli-responsive hydrogel nozzle could
autonomously regulate flow by constricting or widening.
2.4. System construction
To create a system capable of monitoring and responding to stimulus
inputs, the liquid phase construction techniques outlined above are performed in
a wide, shallow cavity with a transparent top and pre-configured external
connections [10]. Rigid polymers form fluidic channels that link with the
external connections. Stimuli-responsive hydrogels act as sensors and valves,
directing flow through the channels and signaling the occurrence of specific
events. Multiple structures of the same chemistry can be polymerized
simultaneously.
while structures of different materials are polymerized sequentially,
flushing the cavity after each polymerization.
c d
Fig. 4. Several examples of standard cavities with preconfigured external connections and
polymerized structures: a) a polycarbonite gasket on a glass coverslide forms a
cavity. A pipette tip fills the cavity with monomer solution, b) A multichannel
single layer channel network, c) A single layer device with connectors, d) Close
up view of a three-layer device. Polymerized channels filled with dye for
visualization purposes in b-d.^im. (D. J. Beebe et al, PNAS 97 (25), 2000 - with
permission [10])
The only requirement of the construction platform is that one surface of the
cavity be transparent to the light required for polymerization. This allows many
different material combinations to be utilized, depending on the required
characteristics of the system. Cavities made of a variety of materials have been
used. The most convenient utilizes a commercially available polycarbonate film
attached to a glass surface with an adhesive gasket (Grace Biolabs, Bend, OR)
as shown in Figure 4. The platform allows for a wide variety of channel
geometries to be formed rapidly. With a common platform and the relatively
short construction times required for polymerization, many types of systems are
able to be created rapidly by polymerizing hydrogel components in different
configurations (Figure 5). The curved structures of section 2.3 can be
incorporated into this construction framework by first polymerizing the curved
structures, and then sealing the transparent top and polymerizing hydrogels with
photo masks as in section 2.2. Ultimately, external computerized delivery of
monomer mixtures and a light array (in place of the photo mask) could allow a
system with a desired functionality to be conceived and created on demand, with
httle required input from the end user.
a b
Fig. 5. A combination of responsive and non-responsive materials are combined using
sequential polymerization to form a check valve, a) The valve immediately after
polymerization, but before the responsive portion is expanded, b) Via exposure to
appropriate fluid environment, the responsive portion of the valve swells creating a
check valve that allows flow in one direction (left to right) and restricts flow in the
opposite direction.
3. DESIGN CONCEPTS
3.1. Introduction
Because the three-dimensional network of a hydrogel is able to retain
solvents, certain species (e.g., ions) dissolved in the surrounding solvent will
penetrate the hydrogel. In some applications, the hydrogel may need to be
isolated from these species, particularly if an unwanted change in volume would
be induced. To address this issue and control the environment inside hydrogel
structures, we have coated hydrogels with fatty acid layers and physically
isolated them with flexible membranes. In other applications, the rate at which
the species penetrates the hydrogel or the rate at which the hydrogel changes
volume may need to be controlled. This control can be achieved by fabricating
composite structures of responsive and inert hydrogels or responsive hydrogels
and rigid polymers.
Also, since the hydrogels are polymerized from a liquid mixture and can be
influenced by the flow of solvents around them, fluid flow characteristics of the
microscale need to be considered. Microscale fluid flow is laminar. Laminar
flow is a smooth, steady flow, without the disturbances seen in turbulent flow.
Two distinct fluids can flow side by side without mixing as they would at larger
length scales. The only mixing that will occur is solely due to diffusion.
10
These properties allow the precise delivery of fluids to a specific spatial
location, for example, exposing only a portion of a hydrogel to a certain input.
In addition, controlled diffusion of the liquid monomer solution prior to
polymerization enables the production of non-homogeneous hydrogels with
spatially varying characteristics.
3.2. Multiple structures and physical supports
If the rate-limiting step in the hydrogel response is the rate at which the
stimulus penetrates the hydrogel, the overall time response will be improved by
shortening the length that the stimulus has to travel. One way to accomplish this
is to fabricate hydrogels with high aspect ratios. However, the pH-sensitive
hydrogels of section 2.2 that have aspect ratios of 1 or more have been found to
buckle and move during volume size changes. In order to create physically
robust hydrogels with small length scales and high aspect ratios, we have
polymerized pH-sensitive hydrogels around rigid polymer support posts as in
Figure 6a. This approach produces hydrogel length scales that are a fraction of
the smallest cavity dimension.
While an individual hydrogel polymerized around a support post has a
small actuation length, an array of these structures will have an effective
actuation length similar to a large hydrogel. The step response for expansion of
the array valve design shown in Figure 6b is 8 seconds (the contraction step
response is of the same order). In contrast, an alternative valve design that uses a
single larger cylindrical structure in the same size channel has a step response of
130 seconds over the same pH range [9]. In this case, the post design accelerates
the response time by a factor of 16, while retaining the actuation length and
functionality of a larger structure (Figure 6c).
3.3. Isolation Schemes
In a system containing numerous differing stimuli, methods must be
employed to ensure that the proper stimulus influences the correct hydrogel. A
nonporous membrane is one such method. If the membrane is flexible, the
hydrogel will be able to interact with other portions of the system by deforming
the membrane. For example, the deformation can throttle the fluid flow in an
adjacent channel. Such a case is shown in Figure 7, where the membrane has
been fabricated from the same elastomeric material (PDMS) that was used to
build the construction platform [13]. This scheme allows the stimuli triggering
the hydrogel to be separated from the environment on the other side of the
membrane.
Another possibility for isolating hydrogels is through the use of fatty acid
coatings [14, 15]. Because a fatty acid coating is able to selectively exclude
certain species (for example, polar solvents and ions), a different environment
can be maintained within the hydrogel interior than in the surrounding