Table Of ContentLaboratory Methods in
Microfluidics
Laboratory Methods in
Microfluidics
Basant Giri
Center for Analytical Sciences,
Kathmandu Institute of Applied Sciences,
Kathmandu, Nepal
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About the Author
Basant Giri received BSc and MSc degrees in Chemistry from Tribhuvan University,
Kathmandu, Nepal, a second MS degree in Chemistry from the Oregon State University,
Corvallis, USA, and a PhD degree in Chemistry from the University of Wyoming, Laramie,
USA. After working asa research fellow at Nepal Academyof Science and Technology, Nepal
for six months, Dr. Giri cofounded the Kathmandu Institute of Applied Sciences in
Kathmandu, Nepal. Currently he works as a scientist at the Center for Analytical Sciences at
the same institute. His research interests include development of low-cost analytical devices
(e.g.,papermicrofluidics)forbiologicalandenvironmentalapplications.Hehasseveralyears
of teaching experience at high school, undergraduate, and graduate levels in Nepal and the
United States as faculty and teaching assistant, respectively. Dr. Giri has authored and
coauthored a textbook on Nanoscience and Nanotechnology and several peer-reviewed
researcharticles.
xiii
Preface
Considering the increasing interest in microfluidics, Laboratory Methods in Microfluidics
aimstofilltheneedforalaboratorybookinthisfield.
Microfluidics is becoming an increasingly popular subject both in education and
research. Many universities are now incorporating microfluidics in their courses to a greater
or lesser extentalong with experiments in the laboratorycourses.Even though there are sev-
eral textbooks covering this topic, there is currently no resource covering experimental pro-
cedures. This laboratory book aims to provide a number of detailed instructions for
experimentsinmicrofluidicsintendedforundergraduateandpostgraduatecoursesinanalyt-
ical chemistry, biochemistry, microbiology, biotechnology, environmental science, and engi-
neering. Some experiments can even be implemented in high-school curriculum projects
andexperiments.
Most of the experiments described in this book have been adapted from research articles
and the experience of the author while teaching undergraduate analytical chemistry labs.
While care has been taken to ensure that the information in this book is correct, neither the
author nor the publisher can accept responsibility for the outcome of the experimental pro-
cedures outlined in this book if not properly followed. The main aim of the book is to serve
as an educational tool to prepare today’s students for the more demanding regimen of
microfluidics. The experiments aimtoprovide practicalexperiencein the application ofclas-
sical and instrumental techniques incorporated in microfluidics. Each experiment includes
background information including learning objectives and an overview of the principles
behind the experiment, a list of materials and chemicals required, safety notes, step-by-step
procedure, additional notes to instructor, assessment questions, and recommendations for
further reading. The instructions for the experiments are so detailed that the measurements
can, for the most part, be taken without the help of additional literature. With Laboratory
Methods in Microfluidics instructors no longer have to refer to many journals and books to
find the right procedures for their experiments. It is assumed that students are familiar with
basic laboratory techniques and procedures in science before starting experiments described
inthisbook.However,somebasicpracticesarecoveredintheAppendix.
In conclusion, this book is a work in progress, and I encourage readers to submit ideas,
suggestions, and comments for improvements or for new experiments. I hope you find this
laboratory manualhelpfulinyourstudy.
xv
xvi Preface
Key features
(cid:129) 18Standalone fine-tunedexperiments
(cid:129) Emphasizesfabricationofmicrofluidic devicesandtheirandapplications
(cid:129) Experimentsusingcommonlyfoundmaterialstominimizethecost
(cid:129) Assessment questionsforeachexperiment
(cid:129) Appropriateillustrationsforeachexperiment
(cid:129) Additionalnotesforinstructorsallowingthemtocustomizetheexperiments
(cid:129) Usefulinformationaboutpreparationoflaboratoryreagentsinappendices
BasantGiri
January,2017
Acknowledgments
I am thankful to Dr. Harish Subedi of Western Nebraska Community College, Nebraska, Dr.
Basu Panthi of Trinity University, Texas, and Dr. Lekh Adhikari of Rappahannock
Community College, Virginia for providing input on the initial draft of this book. Likewise, I
am thankful to Dr. Susma Giri (my wife), Mr. Ankit Pandeya, and Mr. Sagar Rayamajhi of
KathmanduInstitute ofAppliedSciences,Nepalforproofreadingthemanuscript.
I am grateful to my PhD advisor Dr. Debashis Dutta from the University of Wyoming,
who introduced me tothefieldofmicrofluidics. Dr. TristanKinde of Sinclair Oil Corporation
(then graduate student at Dutta group) helped me fabricate the glass microfluidic device
during my early days as a PhD student. The lab methods described in this book such as fab-
rication of glass microfluidic device, enzyme assay, and microfluidic separation were initially
developedforaninstrumentalanalysiscoursebyDuttaLabatUniversityofWyoming.
I express my love and gratitude to my father Krishna and mother Dwarika for their love,
support,patience,andsacrifice.
xvii
1
Introduction to Microfluidics
1.1 Background
The field of microfluidics has been gaining popularity in the scientific community since its
jumpstart about three decades ago. This multidisciplinary field has become a unique plat-
form for chemistry, physics, biology, materials science, fluid mechanics, and engineering
disciplines in terms of understanding both fundamentals and applications. Two other terms
related to microfluidics are lab-on-a-chip and micrototal analysis systems, popularly known
as μ-TAS. It is important to learn microfluidic experiments considering their potential in
analytical applications and their advantages over conventional analytical systems.
Incorporating microfluidics in teaching laboratories enables learning opportunities for
undergraduate andgraduate students,evenhighschoolstudentsandindependentresearch-
ers. As microfluidics require less amount of chemicals/reagents and generate less waste,
universitiescouldreduce thecostrelatedtochemicalsandwastedisposal.
1.1.1 What is Microfluidics?
Microfluidics is the science that deals with the precise control and manipulation of small
volumes of fluids in network of microchannels. Generally, micro means one of the following
features: small volumes (μL, nL, pL, fL) and small size leading to low energy consumption
and special microdomain effects. Small size means at least one dimension of the channel
must be in the range of micrometers. The behavior of fluids at microscale can differ from
macroscale behavior. Factors such as surface tension, energy dissipation, and fluidic resis-
tance start to dominate the system at micro level. A microfluidic chip or device contains a
network of microchannels, which are connected to the outside of the channel by inputs and
outputs pierced through the chip. Such connections serve as an interface between the
macro- and microworld. Through these holes, the liquids or gas are injected and removed
from the microfluidic chip. The small size of microfluidic devices offers several advantages
including less sample and reagent consumption, low cost, short analysis time, portability,
etc.1
Microfluidic technologies are not just for education and research. These technologies
have now been incorporated into many commercial products. Inkjet printheads are an
example of the most successful application of microfluidics.2 Printers used to reproduce
digitalimagesproducedbycomputerscommonlyusesuch inkjet printers.Othercommercial
products basedonmicrofluidicsinclude:
1. Agilentbioanalyzer:Thebioanalyzerinstrumentprovidesplatformforbioassays,based
onelectrophoresisandflowcytometry,ofDNA,RNA,proteinsandcellswithlessthan
fourmicrolitersofsample.3
LaboratoryMethodsinMicrofluidics.DOI:http://dx.doi.org/10.1016/B978-0-12-813235-7.00001-5 1
©2017ElsevierInc.Allrightsreserved.
2 LABORATORYMETHODSINMICROFLUIDICS
2. HPLC-Chip/MSsystem:Produced byAgilentthissystemisbasedonmicrofluidicchip
technologyandisdesignedfornanosprayliquidchromatography/massspectrometry
(LC/MS).Accordingtothemanufacturer,thissystemisrobust,reliable,sensitive,and
easytouseforbiomarkerdiscoveryandvalidation,monoclonal antibody
characterization,small-moleculeanalysis,phosphopeptideanalysis,etc.4
3. Caliper LabChip platforms: Caliper of the PerkinElmer company has produced a
number of LabChip devices/kits5 based on microfluidics involving both
electrokinetic and pressure-driven flows. These devices can be used in bioassays for
drug discovery applications such as small-molecule screening, fragment based
screening, target specificity profiling, etc. The genomic DNA LabChip is used for
DNA analysis.6
4. Point-of-carebloodanalyzersandothermedicaldiagnosticplatformsfromcompanies
including7Abaxis,Abbott,Achiralabs,Biosite,Biovitesse,Biolithic,Baebies,Boston
Microfluidics,CardioMEMS,GenePOC,FluidMedix,Micro2Gen,Nanosphere,
Nanomix,etc.
1.2 Frequently Used Microfluidic Terms
1.2.1 Laminar Flow
One of the important properties of fluid flow in the microdomain is laminar flow. In laminar
or streamline flow, fluids flow side-by-side in parallel layers and do not necessarily mix
unlike in turbulent type flow. Adjacent layers slide past one another like playing cards. The
only mixing in laminar flow is through diffusion. Laminar flow is characterized by high-
momentumdiffusionandlow-momentum convection.Innonscientificterms,laminar flowis
smooth while turbulent flow is rough. Flow of water in steep river is an example of turbulent
flow. Type of flow is characterized by a dimensionless parameter known as the Reynolds
number (R ). In microfluidic systems, the R is usually less than 100 and the flow is consid-
e e
eredaslaminarflow8(Fig.1.1).
FIGURE1.1 Schematicsoflaminarflowandturbulentflow.
Chapter1(cid:129)IntroductiontoMicrofluidics 3
1.2.2 Electroosmosis and Electrophoresis
These two phenomena occur when electric field is applied across the terminals of a micro-
channelcontainingfluid.
When electric field is applied across the two terminals of a microchannel, the bulk of the
liquid inside the channel moves from one pole to the other. This motion of fluid is called
electroosmotic flow (EOF), synonymously called electroosmosis (see Fig. 1.2). The velocity of
thisfluidflowdependsontheappliedvoltage,microchannelmaterial,andnatureofthefluid
itself. When polar liquid such as water is brought into contact into the surface of the micro-
channel, the microchannel surface acquires an electric charge with a thin layer of charges
very close to the surface, which is known as electric double layer. When an electric field is
applied to the fluid via electrodes placed at inlets and outlets, the net charge in the double
layeris induced tomove bytheresultingCoulombforce. Inanegatively charged microchan-
nel surface, the EOF is directed toward the negatively charged cathode through the
microchannel.8
The EOF velocity (ν ) in cm/s depends on applied electric field (E) and can be calcu-
EOF
lated using Eq. (1.1). Electric field in V/cm is the applied voltage per unit length of the
channel.
ν 5E(cid:1)μ ð1:1Þ
EOF EOF
where μ is the electroosmotic mobility, which depends on the device material and buffer
EOF
solutionandisdefinedby:
Eζ
μ 5 ð1:2Þ
EOF η
where ζ is the zeta potential of the channel wall, E is the relative permittivity of the buffer
solution, and η is the viscosity of the fluid. Experimentally, ν can be determined by mea-
EOF
suring the retention time of a neutral analyte in a channel (see Chapter 5: Determination of
FIGURE1.2 (A)AschematicdescribingEOFandEP.Whenelectricfieldisappliedacrossthetwoterminalsofa
channel,negativelychargedanalytesmigratetowardanodewithEPandbulkfluidmovingtowardcathodewith
EOF.ThenetflowvelocityoftheanalyteisacompromisebetweenEOFandEP.(B)Variousforcesactingupona
chargedparticleundertheinfluenceofappliedelectricfield.
