lab on a chip handout
TRANSCRIPT
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ADVANCED BIOENGINEERING METHODS LABORATORY
MICROFLUIDICS LAB ON CHIP
Aleksandra Radenovic
1
MICROFLUIDICS LAB ON CHIP
ABSTRACT
Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative
description of the dynamics of many important cellular processes. Such a description will require novelexperimental techniques that enable the generation of time series data for the governing regulatory proteins in a
large number of individual living cells. An ideal data acquisition system would allow for the growth of a large
population of cells in a defined environment which can be monitored by high resolution microscopy for anextended period of time. Thus this lab will consist on a brief theory about microfluidics then will follow the
practical work going from the chip fabrication to one of its applications: the tracking or monitoring of particles
(beads or E. coli) in this device and the subsequent analysis of the acquired data. Some imaging techniques will
also be introduced. Finally, a few questions will be discussed in order to outline some important points.
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ADVANCED BIOENGINEERING METHODS LABORATORY
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TABLE OF CONTENTS
1 Theory ............................................................................................................................................. 3
1.1 Basic Principles of Microfluidics ............................................................................................ 4
1.2 Device Fabrication.................................................................................................................. 5
1.3 Questions (Theory) .................................................................................................................. 8
1.4 Integration of Microfluidics and Microscopy .......................................................................... 9
2 Practical work .............................................................................................................................. 11
2.1 Material requirements ............................................................................................................ 11
2.2 PDMS silicon mold ............................................................................................................... 11
2.3 Integration of Microfluidics and Microscopy ........................................................................ 14
2.4 Run the sample ...................................................................................................................... 16
2.5 Viewing / Tracking Particles in Device Geometry ................................................................ 17
2.6 Epifluorescence ..................................................................................................................... 18
3 Data analysis ................................................................................................................................ 19
3.1 Calibration ............................................................................................................................. 193.2 Particle Tracking ................................................................................................................... 19
3.3 Matlab analysis ...................................................................................................................... 23
3.4 Particle analysis ..................................................................................................................... 23
3.5 Questions ............................................................................................................................... 26
4 References .................................................................................................................................... 26
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ADVANCED BIOENGINEERING METHODS LABORATORY
MICROFLUIDICS LAB ON CHIP
Aleksandra Radenovic
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1. THEORY
Recent progress in reconstructing gene regulatory networks has established a framework for a quantitative
description of the dynamics of many important cellular processes. Such a description will require novelexperimental techniques that enable the generation of time series data for the governing regulatory proteins in a
large number of individual living cells. An ideal data acquisition system would allow for the growth of a large
population of cells in a defined environment which can be monitored by high resolution microscopy for anextended period of time.
In this laboratory exercise we fabricate and use such a data acquisition system. With our setup, the gene
expression state of each cell could be monitored for the length of the experiment, giving the experimenter
accurate data about the temporal progression of each individual cell within the larger population. To this end,
bioengineers have increasingly used devices with fluid channels on the micron scale known as microfluidic
devices. The goal of this exercise is to fabricate and use such a microfluidic device.
Figure 1. Microfluidics provide a tool for the miniaturization and serial processing of fluids allowing better control of their
properties, integration of different operations and parallelization. Reproduced from1
Microtechnology in general, and microfluidics in particular, can facilitate the accurate study of cellular behaviorin vitro because it provides the necessary tools for recreating in vivo-like cellular microenvironments.
Microfluidics involvethe handling and manipulation of very small fluid volumes, enabling creation and control ofmicroliter-volume reactors while drawing advantages from low thermal mass, efficient mass transport, and large
surface area-to-volume ratios.
Because fluid viscosity, not inertia, dominates fluid behavior at this scale, microfluidic flow is laminar, ensuringthat the system does not include turbulent flows which would be detrimental for observing cellular behavior under
high magnification.Lately, microfluidic lab-on-a-chip devices have become increasingly valuable as the known complexity of
gene networks grows, driving the need for reduced-scale assays in probing entire parameter spaces of genetic
circuits. The result has been the development of integrated microfluidic circuits analogous to their electricalcounterparts, which aim to support large-scale multi-parameter analysis in parallel. Recent applications ofmicrofluidics in biotechnology include DNA amplification, purification, separation 2, and sequencing3; large-scaleproteomic analysis
4; development of memory storage devices
5; cell sorting
6and single-cell gene expression
profiling.The use of microfluidic devices to conduct biomedical research and create clinically useful technologies has a
number of significant advantages. First, because the volume of fluids within these channels is very small, usually
several nanoliters, the amount of reagents and analytes used is quite small. This is especially significant for
expensive reagents. The fabrication techniques used to construct microfluidic devices (discussed in more depth
later) are relatively inexpensive and very amenable both to: highly elaborate multiplexed devices and mass
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ADVANCED BIOENGINEERING METHODS LABORATORY
MICROFLUIDICS LAB ON CHIP
Aleksandra Radenovic
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production. In a manner similar to that for microelectronics, microfluidic technologies enable the fabrication of
highly integrated devices for performing several different functions on the same substrate chip. One of the long
term goals in the field of microfluidics is to create integrated, portable clinical diagnostic devices for home and
bedside use, thereby eliminating time consuming laboratory analysis procedures.
1.1 Basic Principles of Microfluidics
1.1.1 Reynolds numberThe flow of a fluid through a microfluidic channel can be characterized by the Reynolds number, defined as
equation (1.1)
avg
e
L VR
(1.1)
Where L is the most relevant length scale, is the viscosity, is the fluid density, and Vavg is the average
velocity of the flow. For many microchannels, L is equal to 4A/P where A is the cross sectional area of the
channel and P is the wetted perimeter of the channel.
Due to the small dimensions of microchannels, the Re is usually much less than 100, often less than 1. In this
low Reynolds number regime, flow is completely laminar and no turbulence occurs the transition to turbulentflow generally occurs in the range of Reynolds number 2000. Laminar flow provides a means by which molecules
can be transported in a relatively predictable manner through microchannels. Note, however, that even at
Reynolds numbers below 100, it is possible to have momentum-based phenomena such as flow separation.
1.1.2Poiseuilles Law
In such a laminar flow of viscous and incompressible fluid, the pressure drop and the flow rate, as well as theeffective resistance might be obtained by using the Poiseuille equation (1.2).
and (1.2)
Where p is the pressure drop, Q is the volumic flow rate, R is the resistance to flow, L is the length of thechannel, r radius of the channel, is the dynamic fluid viscosity and x the distance in direction of flow.
1.1.3 Pressure Driven Flow
There are two common methods by which fluid actuation through microchannels can be achieved. In pressuredriven flow, the fluid is pumped through the device via positive displacement pumps, such as syringe pumps.
One of the basic laws of fluid mechanics for pressure driven laminar flow, the so-called no-slip boundary
condition, states that the fluid velocity at the walls must be zero. This produces a parabolic velocity profile within
the channel (Figure 2.a).The parabolic velocity profile has significant implications for the distribution of molecules transported within a
channel. Pressure driven flow can be a relatively inexpensive and quite reproducible approach to pumping fluids
through microdevices. With the increasing efforts at developing functional micropumps, pressure driven flow is
also amenable to miniaturization. (Figure 2.a)
p=8LQ
r4
R=8x
r4
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ADVANCED BIOENGINEERING METHODS LABORATORY
MICROFLUIDICS LAB ON CHIP
Aleksandra Radenovic
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1.1.4 Electrokinetic FlowAnother common technique for pumping fluids is that of electroosmotic pumping. If the walls of a microchannel
have an electric charge, as most surfaces do, an electric double layer of counter ions will form at the walls. When
an electric field is applied across the channel, the ions in the double layer move towards the electrode of oppositepolarity. This creates motion of the fluid near the walls and transfers via viscous forces into convective motion of
the bulk fluid. If the channel is open at the electrodes, as is most often the case, the velocity profile is uniform
across the entire width of the channel (Figure 2.b). However, if the electric field is applied across a closed channel
(or a backpressure exists that just counters that produced by the pump), a recirculation pattern forms in which
fluid along the center of the channel moves in a direction opposite to that at the walls (Figure 2.c). In closed
channels, the velocity along the centerline of the channel is 50% of the velocity at the walls.
a b
c
Figure 2 a) Velocity profile in a microchannel with aspect ratio 2:5 under conditions of pressure driven flow. Note that the
velocity is assumed to be zero at the walls in most treatments of transport of liquids. b) The very uninteresting flow velocityprofile calculated for electroosmotic pumping in an open channel. Such a channel (in the absence of backpressure) exhibits plugflow. Shown in the situation for negatively charged walls; the anode is at the left and the cathode is at the right. In fact the profileis very interesting close to the walls, since velocity drops to zero at the walls over a distance that is comparable to the thickness
of the electrical double layer. c) The view of the electroosmotic flow velocity vectors in a closed channel. Note that the
recirculation results in equal total flows to the right and left at all vertical planes through the channel. The anode is on the left andthe cathode is on the right and the walls are negatively charged.
1.2 Device Fabrication
1.2.1 Photolithography
In recent years, soft lithography has become the preferred method for fabricating microfluidic devices forbiology. Soft lithography includes a suite of methods for replicating a pattern using elastomeric polymers (Figure
4). Soft lithography can be represented as a three-step process comprised of concept developing, rapid
prototyping, and replica molding.
The first step, concept developing, involves drafting a device design in a computer-aided design (CAD) program.Here, a general idea for a device that serves some purpose is fleshed out using engineering approaches. Using thelaws of fluid dynamics under the condition of low Reynolds number for microvolume flow, fluid channelresistances are calculated and modified to satisfy desired driving pressures and flow rates. Following fine-tuning
of the entire channel architecture, the device design is broken up into multiple layers, where all features of a given
height are placed on a single layer for photolithographic purposes. Finally, all the device layers are printed at high
resolution onto transparency film. These are then fastened to ultra -transmissive borosilicate glass for use as aphotomask set in the following contact lithography step. Or alternatively, as we did in the CMI (Center of
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ADVANCED BIOENGINEERING METHODS LABORATORY
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MicroNanoTechnology EPFL) for this laboratory, a photomask may be created by patterning chrome on a glass
plate.
For the design we have chosen the recently proposed microfluidics chip that has been used for monitoring the
collective synchronization properties in an engineered gene network with global intercellular coupling in agrowing population of cells that exhibit spatiotemporal waves occurring at millimeter scales
7. The chip design is
shown in Figure 3.
Figure 3. Microfluidic device used for maintaining E. coli or beads at a constant density. The main channel (blue) supplies mediato cells in the trapping chamber, and the flow rate can be externally controlled to change the effective rates of an engineered
gene network7.
There is presented the lithography concept to understand the how have been made the wafer that you will use tofabricate your device. Due to the time constraints of this exercise, this part of fabrication is already made by TA.
In rapid prototyping, a positive or negative photoresist is spin coated onto a clean silicon wafer at a speci fied
thickness and then exposed to UV light through the photomask to selectively crosslink the features represented by
the mask. Since each exposure iteration creates all device features of a given height (being the depth of the
photoresist layer); this process can be repeated to pattern the wafer for multi-layer device features. The final result
is a positive relief of photoresist on the silicon wafer, known as a master mold, whose topology preciselyreflects the desired device channel and feature structures and can be used repeatedly to form successive batches of
devices. Fabrication of this master mold completes the rapid prototyping step of soft lithography. The final step,called replica molding, involves the casting of a transparent, silicone-based liquid prepolymer (usually PDMS)
against the master mold to generate a negative replica of the master.
The prepolymer is first poured onto the wafer and heat-cured in place to form a rubbery silicone solid. This
silicone monolith is then peeled from the mold to reveal the inverted feature topology represented by the mold.For example, ridges on the master mold appear as valleys in the replica. This monolith is then diced into
individual devices, bored with a cylindrical punch to form holes for connection to fluid reservoirs, and cleanedusing Scotch tape and methanol. In the final step, the feature sides of the devices, along with opposing coverslip
surfaces, are briefly treated with low power oxygen plasma. This process activates the surfaces of the PDMSdevices and glass coverslips so that they form a permanent bond when placed in contact. In bonding the two
objects, fluid channels in the PDMS are sealed against the flat coverslip surface to form microchannels internallyconnecting the device fluidic ports. These finished devices mark completion of the replica molding step of softlithography
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ADVANCED BIOENGINEERING METHODS LABORATORY
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or create chrome mask
Your TAs prepared wafers beforehand
Concept copied formDanino et al. Nature 463, 326-330 (2010)
Your will fabricate microfluidic devicefrom this point !
Figure 4. Schematic of microfluidic device fabrication using soft lithography (adapted from Ref.8)
1.2.2 Multilayer soft lithography
The techniques described here can be extended to perform multilayer soft lithography, which provides the
capability to bond multiple patterned layers of elastomer to create active microfluidic systems containing on -off
valves, switching valves, and pumps. There will not be any such sophisticated components in our device but, it is
always good to know for your future research since it is more and more used in laboratories. In multilayer soft
lithography, in addition to a layer of microchannels cast in PDMS as described before, a second, deformable thin
membrane of PDMS is cast by spin coating PDMS onto a master mold. This allows for a layer with a thickness of
only about 50 to 100 microns. The thin PDMS layer is then partially cured, and bonded to the thick PDMS layer.
Both layers are then bonded to a flat substrate.
Figure 5. a) Multilayer soft lithography fabrication process. A microchannel layer is molded in a thin deformable PDMS membrane
through a spin-coating process. A second layer of microchannels is molded from a thick layer of PDMS. The two PDMS layers are bondedtogether, and the structure is then bonded to a flat substrate. b) Example of a peristaltic pump fabricated from multilayer soft lithography.By successive pressurization of the upper control layer channels, fluid is pumped through the lower fluidic layer. (Adapted from Ref. 1)
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The end result is a set of microchannel layers separated vertically by a thin membrane of PDMS. The advantage
of this architecture is that air or fluid pressure in one of the microchannels can be used to deform the membrane,
blocking or constricting fluid flow in the second microchannel. This allows for simple integration of valves and
pumps into these multilayered fluidic structures. An overview of the fabrication process and an example of aperistaltic pump are illustrated in Fig. 5.
Recent research in the microfluidics field has produced several examples of complex devices with hugely parallelactive channel structures for high-throughput cell analysis. In approaching years, the fundamental benefits of softlithography for biology, which include ease of fabrication, inexpensive production, and rapid device turnover,
will continue to aid the researcher seeking increasingly functional cell assays.
1.3 Questions (Theory)
Q1. How does the laminar flow help microfluidic design? Why?
Q2. Which network has equal flow through branches?
Why? How is it designed in your chip?
Q3. Which path will have higher flow? Why?
Q4. For what are the hooks between media input and waste outputs useful?
Q5. Why do we have 2 inlets and 2 outlets?
Q6. Define low Reynolds number. Typical E.coli (2.0m long and 0.5m in diameter) is characterized
by low or high Reynolds number?
Q7. How are fluidic resistance and channel width related?
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ADVANCED BIOENGINEERING METHODS LABORATORY
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Aleksandra Radenovic
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1.4 Integration of Microfluidics and Microscopy
Microfluidics has recently found wide applications in research aimed at observing cellular development within
dynamic microenvironments. Devices designed for these purposes frequently possess the ability to generatethermal and/or chemical gradients across the cell development volume. Another recently demonstrated strength of
microfluidics is the ability to generate large-scale and highly parallel integrated circuits of fluidic channels forhigh-throughput cellular analysis
11-13. However, for researchers interested in studying the behavior of synthetic
gene circuits, the most challenging goal of microfluidics has been in supporting long-term single-cell analysisfor large sample populations. Therefore, much recent research has focused on this goal using various design
strategies.
One group approached the difficulties in single-cell analysis by developing a microfluidic network enabling thepassive and gentle separation of a single cell from bulk suspension
14. This individual cell is focused by
hydrostatic pressure and laminar flow streams to a trapping region, where integrated valves and pumps enable the
precise delivery of nanoliter volumes of reagents to that cell. Whereas this research focused on individual cells
over a relatively short time span, another group developed a microfluidic platform for long-term cell culturestudies spanning the entire differentiation process of mammalian cells
15. They demonstrated operation of this
device by observing a culture of muscle cells differentiating from myoblasts to myotubes over the course of two
weeks.
To researchers interested in long-term gene expression variability within single-celled prokaryotic and
eukaryotic populations, a chemostat likely represents the ideal cell assay. In recent years, the many challenges
involved in operating continuous macroscale bioreactors (such as the need for large quantities of reagents) have
driven the miniaturization of these devices into microfluidic chip-based formats. In continually providing freshnutrients and removing cellular waste to support exponential growth, the microfluidic chemostat (small cell
trapping region) presents a nearly constant environment that is ideal for long-term cell culture monitoring with
single-cell resolution. Recently, one group presented a microfluidic chemostat for culturing bacterial and yeast
cells in an array of shallow microscopic chambers with support for dynamically-defined media16. Similarly, arecent implementation of a microfluidic bioreactor has enabled long-term culturing and monitoring of smallpopulations of bacteria with single-cell resolution17. This microchemostat contained an integrated peristaltic pump
and a series of micromechanical valves to add medium, remove waste, and recover cells. The device was used to
observe the dynamics of an E. coli strain carrying a synthetic population control circuit that regulates celldensity through a feedback mechanism based on quorum sensing.
A final implementation of the chemostat design was utilized to precisely control and constrain exponentialgrowth of the yeast S.cerevisiae and E. coli to a monolayer
18. Here, dimensions of the chemostat device were
precisely controlled to constrain exponential growth of yeast and E. coli cells to a monolayer. The device has
been modified for imaging a culture of cells growing in exponential phase for many generations. The construction
was such that a shallow trapping region will constrain a population of cells to the same focal plane.
The significant advantage of monolayer growth in a height-constrained chamber was demonstrated by
visualization of a group of cells residing at the trapping region boundary. Through directed planar growth, the
researchers were able to resolve the temporal evolution of single-cell gene expression levels with the aid of
segmentation and tracking software. Advantages of this device design and software package included simple
operation and automated single-cell fluorescence trajectory extraction. Such novel data should prove useful in
investigating the timing and variability of gene expression within various synthetic gene regulatory network
architectures on the time scale of many cellular generations.
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1.4.1 Fluorescence imaging
Fluorescence microscopy is the most popular method for studying the dynamic behavior exhibited in live cell
imaging. This stems from its ability to isolate individual proteins with a high degree of specificity from non-fluorescing material. The sensitivity is high enough to detect as few as 50 molecules per cubic micrometer.
Different molecules can now be stained with different colors, allowing multiple types of molecule to be tracked
simultaneously. These factors combine give fluorescence microscopy a clear advantage over other optical
imaging techniques, for both in vitro and in vivo imaging.
Fluorescence microscope is a light microscope used to study properties of organic or inorganic substances using
the phenomena of fluorescence instead of, or in addition to, reflection and absorption. In most cases, a
component of interest in the specimen is specifically labeled with a fluorescent molecule called a fluorophore
(such as GFP, Green Fluorescent Protein). GFP is a fluorescent protein that was first found in the jellyfish
Aequorea Victoria. It has the useful property that its formation is not species specific. This means that it can be
fused to virtually any target protein by genetically encoding its cDNA as a fusion with the cDNA of the target
protein. This can be done in a live cell, and hence the movement of individual cellular components can now be
analyzed across time.
a) b)
Wavelenght (nm)
Spectrum
Figure 6. a)Fluorescence imaging principle (Wikipedia: Fluorescence_microscopy) b) Excitation and emission spectra of the dyes
used in this practical FITC very close the GFP excitation and emission spectra
There is no requirement to fix and permeablize the cells first. The discovery of GFP has made the imaging of
real-time dynamic processes commonplace, and caused a revolution in optical imaging. The GFP revolution goes
even further with the development of different colored GFP isoforms, such as yellow GFP and cyan GFP. This
allows multiple proteins to be viewed simultaneously in a cell.
In this practical we use 2.5 m PeakFlow green flow cytometry reference beads that stained with fluorescentdye (FITC) that have been carefully selected to produce emission peaks coincident with labeled cells used in
typical flow cytometry applications (GFP labeled cells). Because PeakFlow beads are highly uniform with
respect to both size and fluorescence intensity, and because they approximate the size, emission wavelength and
intensity of many biological samples, they can be used to calibrate a flow cytometers laser source, optics, streamflow and cell sorting system without wasting valuable and sensitive experimental material.
The specimen is illuminated with light of a specific wavelength which is absorbed by the fluorophores, causing
them to emit longer wavelengths of light (of a different color than the absorbed light). The illumination light is
separated from the much weaker emitted fluorescence through the use of a dichroic mirror. Typical componentsof a fluorescence microscope are the light source (Xenon or Mercury arc-discharge lamp), the excitation filter, the
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ADVANCED BIOENGINEERING METHODS LABORATORY
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dichroic mirror (or dichromatic beamsplitter), and the emission filter. The filters and the dichroic are chosen to
match the spectral excitation and emission characteristics of the fluorophore used to label the specimen.
1.4.2FiltersFilters used in this work are called FITC (Figure 7) according to the traditional fluorochromes that were earlier
commonly used for green and red fluorescence. In the figure, the blue (1) curve shows the excitation i.e. the
wavelengths that illuminate the sample. The red (2) curve shows the emission i.e. the wavelengths that are shown
to the viewer.
Figure 7. FITC filter spectrum. 1 = excitation band, 2 = emission band
2. PRACTICAL WORK
2.1Material requirements Handling. Safety glasses, gloves, tweezers, Petri dishes, pipettes, spoons, cups, razor blades, scalpels,
aluminum foil.
Machines. Ventilated fume hood, high precision scale, nitrogen gun, mechanical mixer, vacuumdesiccators, manual hole-punching machine, binocular, oven/hot plate, oxygen plasma.
Products. Sylgard 184 silicone base, Sylgard curing agent, silanizing agent (TMCS:Chlorotrimethylsilane, 33014 from sigma)
2.2PDMS silicon moldsThe first step in PDMS molding is designing molds and creating them. SU-8 processing and silicon etching arethe two protocols commonly used to realize molds for PDMS micro-molding. Procedures for creating these molds
are not presented in this present document. Our TAs prepared molds and they are in the AMBL marked waferholder.
2.2.1 Surface conditioningThe surface conditioning of the mold is important to prevent PDMS sticking. A silanization allows passivation
of the surfaces to aid release from PDMS.
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TMCS is corrosive - causes skin burns, harmful in contact with skin also it is armful if swallowed and it
is respiratory irritant. Therefore its handling should be done under the fume hood and you are suggested towear extra pair of gloves.
1) Put on single use additional gloves and operateonly under the fume hood.
2) Place a few drops of TMCS in the small glassreceptacle located in the desiccator (single use
pipettes are available for that purpose).
Note: If TMCS bottle is not in the glass desiccator,
fetch it in the solvent cabinet located on the rightside of the wet bench.
TMCS
Wafer with
PDMS mold
Glassreceptacle
Desiccator
single usepipettes
3) Remove any dust on the surface of the mold using a
nitrogen gun.4) Place the silicon/SU8 mold in this very same desiccator.5) Close the desiccator and place it under vacuum (this
causes the TMCS to evaporate and to form a passivation
layer on the mold surface).
6) Close well the TMCS bottle (use tape also). Fill-in thechemicals follow-up document.
7) When desired time is reached (~15min), vent thedesiccator. DO NOT breath directly above the open
desiccator.
8) Take your mold back, put the TMCS bottle in thedesiccator and put it back under vacuum.
15 min
2.2.2 MixingDegassing
Silicone prepolymer material is very viscous and sticky. Use additional gloves before handling the liquid
PDMS. Aluminum foil is used as a liner for protecting equipment in contact with the (Petri dishes, scale, etc.). A
single use plastic cup is to be used for preparing the PDMS mixture. The plastic cups are compatible with themechanical mixer and their maximum capacity is 50g. This means the total mixture must weigh 50 g maximum.
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5) Use the mechanical mixer to correctlyhomogenize the mixture (Program 1)
o Mixing: 1min @ 2000rpm
o Defoaming: 2min @ 2200rpm
6) Clean the scale well before switching it off andclean the product bottles
2.2.3 PouringSpin coatingPour the PDMS mixture over the passivated mold placed in a
Petri dish or plastic disposable dish. The interior of that dish
should be protected with aluminum foil.
1) Be careful not to create bubbles while pouring themixture (proceed slowly).
2) The mixture is then degassed in the desiccator to removeany remaining entrapped bubbles. If large bubbles form
at the surface, vent vacuum slowly so the mixture does not foam out. Put it back under vacuum until no
bubbles are visible. This also improves the filling of small structures
2.2.4 BakingCuringPDMS can cure without heating in ~24 hours. To decrease cure time,
put the Petri dish in an oven for 1 hour at ~80C. Curing time
depends on temperature and on the thickness of PDMS. After curing,
the wafer is stable and can be stored for months if necessary. To save
time, we provide you an already cured PDMS.
80 C
1) Put an empty and clean single use plastic cup onthe precision scale- Tare the scale so it displays 0.
2) Add the base PDMS (max 40g) and write down thevalue.3) Tare the scale so it displays 0- Using a pipette, add
the catalyst (max 4g) to reach the ratio value 10:1
4) Place the cup in the mixing machine and adjust therevolution balance dial according to the total
weight of the cup.
Caution: adapter weight of 115g to be added to the
weight of your cup
bubbles no bubbles
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binocular
hole punchingmachine
2.2.5 Alignment - DemoldingCreating access ports by punching
After cooling, the PDMS is easily peeled off and cut. Use adequatetools to perform it (tweezers, razor blades).
1) Cut the PDMS into the desired shape. DO NOT damage the devicenetwork!
2) Create access ports using the manual hole punching machine fittedwith a light source and a video camera. Alignment of PDMS
samples with other glass/PDMS/silicon pieces can be done using the
binocular.
2.2.6 Surface activation for bondingPDMS can be bonded to glass, silicon and itself using
oxygen plasma surface activation. PDMS is hydrophobic,
with a low energy and non-reactive surface. It is therefore
difficult to bond it with other surfaces. By exposing
PDMS to oxygen plasma, its surface becomes hydrophilicand more reactive. This results in irreversible bonding
when it contacts glass, silicon, or even another PDMS
piece that was exposed to the same oxygen plasma.
Contact should be made quickly after plasma expositionbecause the PDMS surface will undergo reconstitution toits hydrophobic and non-reactive state within hours. A fine tuning of the oxygen plasma is necessary: a too long
exposure will create too many Si-OH sites resulting in a non-sticking silica layer. A too short exposure will not
create enough Si-OH sites for good bonding. 100 W, 0.3 torr and 6 sec are suggested as parameters. The bonding
is accelerated if a post-bake is then performed.
2.3 Integration of Microfluidics and Microscopy
2.3.1 Set up the pressure controller
The fluid flow through the device is controlled with a pressure controller, rather than a direct control of the flowrate. This means that the actual flow rates will be a function of the tubing length and diameter, the relative heightof the different components, and the pressures. The needed pressures will therefore be slightly different for each
time the experiment is set up.
The MFCS controller needs a 10 minute warm up period. It should be turned on and warming up while the rest of
the components are prepared.
1) Turn on the controller (power switch on the back).2) Open the MFCS_4C software.3) Press the green button on the front of the controllerthe warm up timer should begin counting down.
Plasma
glass coverslip
PDMS
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Pressure channels
Figure 8.MFCS controller and software interface
2.3.2 Set up the tubingWhile the controller is warming up, prepare the inlet and outlet tubing pieces. Minimizing the overall length of
the tubing used will allow for the use of lower pressures and will also help with the flow stability of the system.
1) Use equal length tubing for both inlets and equal length tubing for both outlets.2) Small pieces of steel adaptor tubing are used to couple the inlet and outlet tubing into the device. The
adaptors will press-fit into the 0.02 ID Tygon tubing and also into the cored holes in the PDMS.3) Use the shortest length Tygon tubing that will reach from the device inlets to the sample tubes (fluiwells),
leaving some room to move the device around on the microscope stage.
4) The sample end of the inlet tubing will fit through the ferrule on the top of the sample tube in the pressurecontroller. The ferrule should be screwed down tightly to get the best pressure control. The end of the
tubing should sit near the bottom of the sample tube.5) Use very short pieces of Tygon tubing for the outlets (~10 cm).6) Arrange the end of the outlet tubing so that the flow can spill into a suitable dish, e.g. a petri dish as
shown in Figure 9.
7) Make sure that the sample tubes are kept at the same height as the device.Inlet 1 Media
Inlet 2 Cells/beads
Outlet 1 Outlet 2
Outlet 1 Outlet 2
Inlet 2 Cells/beads
Inlet 1 Media
WASTE container
Figure 9. Tubing connection
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2.3.3 Sample preparation1) Prepare a bead solution by adding some of the bead stock solution into buffer. A good bead concentration
is at least 10L of 1 % bead stock solution per mL of buffer. Shake vigorously beforehand the bead
solution anytime you handle it.
2) Add about 2 mL of bead solution to a sample tube, and attach the sample tube to the appropriate channelin the sample holder.
3) Fill another sample tube with buffer. Attach the sample tube to the appropriate channel in the sampleholder.
4) Make sure all components are sealed tightly.2.3.4 Microscopy
You will use the Olympus IX 81 inverted compound microscope. Please refer to the Microscopy/Koehler/dark
field illumination section of the master handout to perform this step.
Aside from initial calibration and occasional high-power measurements, you will find the 20x objective and dark-
field illumination most useful.
1) Set up the Kogler illumination2) Then, set the dark field illumination
2.4 Run the sampleIn general, the waste outlets should always be at a lower pressure relative to the media and cell inlets. Since the
outlets are not connected to the pressure controller, a positive pressure on the inlets will satisfy this.
1) To control pressures, the direct control button needs to be clicked.2) The pressures in each channel can be controlled with the appropriate slider, or by entering the
value in the requested pressure box. You dont need to change any of the other control
parameters.3) The flow rate through the device to observe particle motion will be much lower than the flow
rate needed to fill the inlet tubing in a reasonable time. The pressure controller has two channels
with a range from 0-25 mBar and two channels with a range from 0-1000 mBar. Therefore, the
inlet tubing should first be connected to the high pressure channels to fill the inlet tubing quickly,and then switched to the low pressure channels.
4) Use channels 3 and 4 to fill the device with buffer. Make sure the tubing from the controller to the sampleholder is connected from the correct channel to the correct sample.
5)
At pressures of around 50100 mBar, filling the device will only take a minute or so.6) While fluid is flowing, inspect the device under the microscope to see if there are a significant amount ofbubbles still in the device. If so, let the buffer run for a while longer at high pressure.
7) Once the device is filled with fluid, turn the requested pressures to 0. You should be able to see beads inthe channel when the fluid motion is stopped.
8) Switch to controlling the pressure through channels 1 and 2 by changing the tubing connections at thecontroller.
9) When running at low pressures to observe particle motion, the pressure difference between the media andcell/waste ports will be rather small to get flow from both into the waste outletsif one is too high, it willcause backflow into the other. The difference between the two is likely to be less than 1 mBar. Final
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working pressures will be in the range of 2 - 10 mBar. At the lower pressures, the fluid motion will be
slow enough to track the particle motion through the main channel. Only a few beads will enter the traps -
most will flow in main section. A good way to observe beads flowing through the traps is to use the 40X
objective focused on a trap, with a higher pressure setting so that more beads are passing per time period.
2.5 Viewing / Tracking Particles in Device GeometryNow that the device has been contacted and flow regulated, and the microscope has been fully configured, we
are now ready to take data.
1) Use 2.5m beads sample to establish the pixel size of your camera2) Turn on the Andor camera3) Open Andor camera software called Solis4) Turn the switch on the microscope to send an image to CCD camera5) Click on the movie camera icon to get a live image from your sample6) Set up the exposure time to 0.05s *by pressing exposure button (Figure 12),7) To take pixel calibration image open in the main menu acquisition, under setup CCD, select c Single
enter following values, exposure time 0.01-0.075s, next under Setup acquisition open binning to 512-512
pixels , you can move binning box to the region around your 2.5m bead press Ok and close Acquisition
menu
8) Press Record and save image as sif file9) Now we are ready to collect movies for your analysis session10) To setup your movies, exposure time t , kinetic series length (number of frames in your movies ) open
in the main menu acquisition, under setup CCD, select Kinetic series enter following values, exposure
time 0.01-0.075s, kinetic series length 500, next under Setup acquisition if necessary open binning to 512-
512 pixels , you can move binning box to the region containing the most beads. Mark in your notebook
the values you entered. Otherwise note in the notebook that you have take full images (1392*1040)
11) Press record12) Save files as sif . Collect all necessary data and save them in your folder13) Repeat this for several bead speeds.14) Once you have finished data collection, you will need to convert all sif. files in raw files. You can do it
file by file or using a batch conversion option in File menu (Main Menu). Make sure that you convert it in
16 bit unsigned integer (with range 0-65322). This is format required for the analysis session.
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RecordLive Exposure
Figure 10. Andor Solis program for data acquisition. Bright field image of device and 1m beads.
2.6 Epifluorescence
Before starting, turn on the Mercury LAMP
controller.
We have only one filter cube set suitable or imagingof FITC labeled beads and GFP labeled bacteria.
Locate its position (out of 6 possible) and open the
filter cube shutter. If you observe bright blue light
as shown on Figure 11, you have located it!
Figure 11. Epifluorescence
1) Now you can close the shutter and put light protection on the microscope body.2) Use brightfield first to locate your specimen.3) Switch to the Mercury lamp as the light source
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4) Open the filter cube shutter5) Make sure that sample is uniformly illuminated (if not open the diaphragm located close to the Mercury
lamp)
6) Repeat the same measurements as in bright filed (for imageJ analysis fluorescence movies are easier toanalyze!) Steps 6-14 are same maybe you will need to adjust exposure timeNote. When not viewing the specimen, close the fluorescence shutter (push the shutter slider in) to minimize
photobleaching of the specimen.
Depending on the objective size you should observe something similar to images shown below!
a) b)
Figure 12. Fluorescence images of the device and beads using in a) 5 X in b) 40 x objective
7) Now stop running beads and try to flush only medium (water through the channel) this should removemost of the beads from channels and leave the one in the traps.
8) Take fluorescence images of 5 traps!3. DATA ANALYSIS
3.1 Calibration (done by TA beforehand)
1) Open in ImageJ your dark filed image of 2.5m beads taken for bin size of512*512 pixels or 1392*1040 pixels.
2) Use a line tool in ImageJ toolbox to draw a line across the selected bead. Belowthe Image J toolbox you will notice x,y coordinates of your line together with
the angle and the length of the drawn line. Make sure to draw the line straight
across the bead diameter.
3) Next open Analyze\Set Scale from File menu where you enter the length of2.5m bead as known distance. It will calculate the pixel aspect ratio of either 1
(512*512) or 1.33 (1392*1040). Use these parameters to set a scale on all
movies that you will be processing.
Make sure you have used same objective and same binning!
3.2 Particle Tracking
To obtain single particle trajectories from recorded movies you will need to use Particle Detector and Tracker
which is an ImageJ Plugin for particles detection and tracking from digital videos.
The plugin implements the feature point detection and tracking algorithm as described in recent publication by
Sbalzarini et al.6This plugin presents an easy-to-use, computationally efficient, two-dimensional, feature point-
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tracking tool for the automated detection and analysis of particle trajectories as recorded by video imaging in cell
biology. The tracking process requires no apriori mathematical modeling of the motion, it is self-initializing, it
discriminates spurious detections, and it can handle temporary occlusion as well as particle appearance and
disappearance from the image region. The plugin is well suited for video imaging in cell biology relying on low-intensity fluorescence microscopy. It allows the user to visualize and analyze the detected particles and foundtrajectories in various ways: i) Preview and save detected particles for separate analysis; ii) Global non
progressive view on all trajectories; iii) Focused progressive view on individually selected trajectory and iv)
Focused progressive view on trajectories in an area of interest.
It also allows the user to find trajectories from uploaded particles position and information text files and then to
plot particles parameters vs. time - along a trajectory.
3.2.1 File opening
1) Before the plugin can be started you must open an image sequence or a movie in ImageJ. For openingyour saved movie use the Import\ Raw from the File menu. You should input following parameters asindicated in Figure 13. (Check your lab notes for number of frames and binning size). Upon file import
you should obtain video sequence of your moving beads.
Figure 13. Import parameters.
2) Next you need to improve contrast and adapt your movie so that it can be treated with ParticleTrackerplugin. To do so use the Image\Type\ 8 bit option from File menu.
3) Next you need to increase contrast you will do it by using Process\Enhance Contrast option from Filemenu. It is safe to select 0.1%saturated pixels under Use Stack Histogram.
4) To filter out noise use Process\Filter\Gaussian blur option from File menu. Again safe sigma value touse is 1.2. Before applying this filtering you can preview your movie.
Q8. What is going on when your sigma is larger than 3?
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3.2.2 Plugin start
5) Now, that the movie is open and compatible with the plugging, you can start the plugin by selectingParticleTracker from the Plugins -\Particle Detector & Tracker menu. After starting the plugin, a dialogscreen is displayed. The dialog has two parts Particle Detection and Particle Linking.
Figure 14. Parameters for better movie quality.
Particle Detection: This part of the dialog allows you to adjust parameters relevant to the particledetection (feature point detection) part of the algorithm
Preview the detected particles: in each frame according to the parameters. This options offersassistance in choosing good values for the parameters. Save the detected particles according to the
parameters for all frames. The parameters relevant for detection are:
Radius: Approximate radius of the particles in the images in units of pixels. The value should beslightly larger than the visible particle radius, but smaller than the smallest inter-particle separation.
Cutoff: The score cut-off for the non-particle discrimination Percentile: The percentile (r) that determines which bright pixels are accepted as Particles. All local
maxima in the upper rth percentile of the image intensity distribution are considered candidate Particles.
Unit: percent (%).
6) Clicking on the Preview Detected button will circle the detected particles in the current frame accordingto the parameters currently set. To view the detected particles in other frames use the slider placed under
the Preview Detected button. You can adjust the parameters and check how it affects the detection by
clicking again on Preview Detected. Depending on the size of your particles and movie quality you willneed to play with parameters.
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Note that very rarely you detect all particles in the field of view mostly due to the fact that they quickly go out of
focus
7) To start on 2.5m beads, enter these parameters: radius = 6, cutoff = 0, percentile = 0.4 and click onpreview detected. Check the detected particles at the next frames by using the slider in the dialog menu.
With radius of 5 they are rightly detected as 2 separate particles. If you have any doubt they are 2 separate
particles you can look at the 3rd frame. Change the radius to 10 and click the preview button. With this
parameter; the algorithm wrongfully detects them as one particle since they are both within the radius of10 pixels.
8) Try other values for the radius parameter. Go back to these parameters: radius = 5, cutoff = 0, percentile =0.4 and click on preview detected. It is obvious that there are more 'real' particles in the image that were
not detected. Notice that the detected particles are much brighter then the ones not detected. Since the
score cut-off is set to zero, we can rightfully assume that increasing the percentile of particle intensity
taken will make the algorithm detect more particles (with lower intensity). The higher the number in the
percentile field - the more particles will be detected. Try setting the percentile value to 2. After clicking
the preview button, you will see that much more particles are detected, in fact too many particles - youwill need to find the right balance (for our dark filed movies between 0.3-0.7 )
There is no right and wrong here - it is possible that the original percentile = 0.1 will be more suitableeven with this film, if for example only very high intensity particles are of interest.
Figure 15. Parameters for particle detection. On the left panel with default values. In the right movie with particlesidentified using following parameters. radius = 5, cutoff = 0, percentile = 0.4
3.2.3 Viewing the results
9) After setting the parameters for the detection (we will go with radius = 5, cutoff = 0, percentile = 0.6) youshould set the particle linking parameters. The parameters relevant for linking are:
Displacement: The maximum number of pixels a particle is allowed to move between two succeedingframes
Link Range: The number of subsequent frames that is taken into account to determine the optimalcorrespondence matching.
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10) These parameters can also be very different from one movie to the other and can also be modified afterviewing the initial results. Put following initial guess for the displacement=5 and link range =3.You can
now go ahead with the linking by clicking OK.
11) After completing the particle tracking, the result window will be displayed. Click the Visualize allTrajectories button to view all the found trajectories.
12) Window displays an overview of all trajectories found. It cannot be saved! It is usually hard to makesense of so much information. One way to reduce the displayed trajectories is to filter short trajectories.
Click on the Filter Options button to filter out trajectories under a given length. Enter 75 and click OK.
(Be careful, if you select to long length you might end up with very few trajectories and lose
information!).
13) Select a trajectory by clicking it once with the mouse left button. A rectangle surrounding the selectedtrajectory appears and the number of this trajectory will be displayed on the trajectory column of the
results window.
14) Now that a specific trajectory is selected, you focus on it to get its information. Click on SelectedTrajectory Info button. The information about this trajectory will be displayed in the results window.
15) Click on the Focus on Selected Trajectory button - a new window with a focused view of this trajectory isdisplayed. This view can be saved with the trajectory animation through the File menu of ImageJ. Look at
the focused view and compare it to the overview window - in the focused view only the selected
trajectory is displayed.
16) Finally you can save the data by pressing Save Full report. Repeat particle tracking for all 3 experimentalconditions measured in the first part of the practical work (2 different speeds).
3.3 Matlab analysis
Now when you have obtained single particle tracks for two different speeds by using provided matlab
code you can:
1) Plot trajectories of certain length (not shorter than 50 frames)2) Calculate speed (mark the exposure time)3) Find and plot MSD
3.4 Particle analysis (If you have enough time)
The goal of this part is to count and determine the size distribution of trapped fluorescent beads.
Particle counting can be done automatically if the specimen lends itself to it, i.e. the individual particles can touch
but not too much! If automatic particle counting cannot be done, ImageJ can facilitate manual counting with the
Point Picker or Cell counter plugin.
3.4.1 Automatic Particle counting
The biggest issue is one referred to as segmentation which is to distinguish the object from the background.
Once the objects have been successfully segmented, they can then be analysed.
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3.4.2 Loading an image into the ImageJ program
1) Open the ImageJ program.2)
Go to File/Open. Select the picture that you would like to analyze.Note: It may be the case that you need to convert the image to a JPEG or analogous format. To do this,
simply open the picture with Preview and save the picture as a JPEG using Save As, then use the toggle to
select the format to JPEG.
3) You should now be able to see the image that you want to analyze.Optional: If you would like to know the actual areas of objects on the screen, say the areas of a collection of cells,
and then knowing units is a must. ImageJ does this with ease:
4) On the ImageJ Tool Bar, select the straight-line icon. (This is on the same tool bar as a square, an oval,etc. Its the same tool bar that opens up with ImageJ.)
5) Using the straight-line tool, use the cursor to mark the length of any object on the picture that you knowthe length of.
6) On the top tool bar, select Analyze.7) Scroll down to Set Scale.8) Fill in Known Distance to the length of the object that you are measuring. This allows ImageJ to set up a
pixel to distance ratio that allows area to be expressed in the appropriate units.
9) Fill in the units of measurement. Any units of distance should work: cm, mm, microns, etc.10) Click Global.11) Click Okay.12) To check that you have indeed set the scale, use the line tool again and measure another object. Select
Analyze and then select Measure. A window should pop up that displays the length of the object you just
measured.
3.4.3 Particle analysis
1) Click on the image in the ImageJ window.2) On the top tool bar select Image. Select Type.3) Select 8-bit. This converts the image into a
format that makes analysis possible. You
should now see that your image is no longer in
color.
4) If necessary crop only image part that contains trapped particles5) You can do that by drawing the rectangle and by cropping the image Ctrl +Shift+X6) On the top tool bar select Image.7) Duplicate Image8) On the top tool bar select Image.9) Select Adjust, the select Threshold.10) This step should have turned all of the objects of interest Red. (adjust scrollbar ) and then clicking on
Apply once you are satisfied with the selection
11) This will turn your image in binary image (if you need you can fill holes by pressing Process-Binary-FillHoles.)
12) If two particles are joined together, you can use processing filters such as Process Binary Watershed toseparate them.
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13) On the top tool bar, select Analyze.14) Select Analyze Particles.15) A window will pop up. Under Size (in the units you specified), give
the area that you want to analyze a lower bound. So if you wanted a
minimum of say 10m2, you would write 10-Infinity in the box. Click
Display Results and dont click any of the other boxes. All otherboxes should be clear of check marks.
16) In the Toggle Menu, select Outlines.17) Click Okay.18) Two windows should have popped up. One with the areas of the
objects listed in the units you specified and another window with the
objects outlined with numbers inside their outlines. Each number
with an area corresponds to the area of the object with that number in
it.
19) Results will be saved as excel file.
3.4.4 Statistics
1) If you would like a distribution of the areas, click on your image again. The objects of interest should stillbe in red.
2) On the top tool bar, select Analyze.3) Select Distribution.4) Unselect Automatic Binning.5) Write in the number of bins that you want and what area range to consider.6) Click Okay.7) A window should come up that gives you all necessary statistics for the areas of the objects in your
picture and their distribution in a bar format.
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3.5 Questions
Q9. What is the average velocity of the moving beads? In which section of the channel does the interface move the
fastest for a given applied pressure? Compute according to the Error Propagation Handout the standard deviation ofthe average velocity.
Q10. For a given applied pressure, how will the fluid speed vary in the differently sized channels (as observable) by
looking at the motion of the beads? Why?
Q11. What is an average number of trapped beads? Can you suggest how to increase the number of trapped beads?
Q12. Propose one biological application for a Lab on the chip device.
4. REFERENCE
1 Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A. & Quake, S. R. Monolithic microfabricated valvesand pumps by multilayer soft lithography. Science288, 113-116 (2000).
2 Ashton, R., Padala, C. & Kane, R. S. Microfluidic separation of DNA. Current Opinion in Biotechnology14, 497-504, doi:Doi 10.1016/S0958-1669(03)00113-7 (2003).
3 Paegel, B. M., Blazej, R. G. & Mathies, R. A. Microfluidic devices for DNA sequencing: samplepreparation and electrophoretic analysis. Current Opinion in Biotechnology 14, 42-50, doi:Doi10.1016/S0958-1669(02)00004-6 (2003).
4 Lion, N. et al. Microfluidic systems in proteomics. Electrophoresis 24, 3533-3562, doi:DOI10.1002/elps.200305629 (2003).
5 Groisman, A., Enzelberger, M. & Quake, S. R. Microfluidic memory and control devices. Science300,955-958 (2003).
6 Huh, D., Gu, W., Kamotani, Y., Grotberg, J. B. & Takayama, S. Microfluidics for flow cytometricanalysis of cells and particles. Physiol Meas26, R73-R98, doi:Doi 10.1088/0967-3334/26/3/R02 (2005).
7 Danino, T., Mondragon-Palomino, O., Tsimring, L. & Hasty, J. A synchronized quorum of geneticclocks.Nature463, 326-330, doi:Doi 10.1038/Nature08753 (2010).
8 Lin, F. et al. Generation of dynamic temporal and spatial concentration gradients using microfluidicdevices.Lab on a Chip4, 164-167, doi:Doi 10.1039/B313600k (2004).
9 Dertinger, S. K. W., Chiu, D. T., Jeon, N. L. & Whitesides, G. M. Generation of gradients havingcomplex shapes using microfluidic networks.Anal Chem73, 1240-1246 (2001).
10 Mao, H., Yang, T. & Cremer, P. S. A microfluidic device with a linear temperature gradient for paralleland combinatorial measurements.J Am Chem Soc124, 4432-4435, doi:ja017625x [pii] (2002).
11 Hong, J. W., Studer, V., Hang, G., Anderson, W. F. & Quake, S. R. A nanoliter-scale nucleic acid
processor with parallel architecture.Nature Biotechnology22, 435-439, doi:Doi 10.1038/Nbt951 (2004).12 Fu, A. Y., Chou, H. P., Spence, C., Arnold, F. H. & Quake, S. R. An integrated microfabricated cell
sorter.Anal Chem74, 2451-2457, doi:Doi 10.1021/Ac0255330 (2002).13 Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. & Leibler, S. Bacterial persistence as a phenotypic
switch. Science305, 1622-1625, doi:DOI 10.1126/science.1099390 (2004).14 Wheeler, A. R. et al. Microfluidic device for single-cell analysis. Anal Chem 75, 3581-3586, doi:Doi
10.1021/Ac0340758 (2003).15 Tourovskaia, A., Figueroa-Masot, X. & Folch, A. Differentiation-on-a-chip: A microfluidic platform for
long-term cell culture studies.Lab on a Chip5, 14-19, doi:Doi 10.1039/B405719h (2005).
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ADVANCED BIOENGINEERING METHODS LABORATORY
MICROFLUIDICS LAB ON CHIP
Aleksandra Radenovic
16 Groisman, A. et al. A microfluidic chemostat for experiments with bacterial and yeast cells. NatureMethods2, 685-689, doi:Doi 10.1038/Nmeth784 (2005).
17 Balagadde, F. K., You, L. C., Hansen, C. L., Arnold, F. H. & Quake, S. R. Long-term monitoring of
bacteria undergoing programmed population control in a microchemostat. Science309, 137-140, doi:DOI10.1126/science.1109173 (2005).18 Cookson, S., Ostroff, N., Pang, W. L., Volfson, D. & Hasty, J. Monitoring dynamics of single-cell gene
expression over multiple cell cycles. Mol Syst Biol, -, doi:Artn 2005.0024 Doi 10.1038/Msb4100032(2005).