A Project Report

On
Applications of Droplet Based Microfluidics
BY
SAMARPITH S ROUT
2020AAPS1332H

Under the supervision of

PROF. SANKET GOEL

SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF

BIO F226: STUDY PROJECT

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE PILANI (RAJASTHAN)

HYDERABAD CAMPUS
(DECEMBER 2023)

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 ACKNOWLEDGEMENTS

I sincerely thank Prof. Sanket Goel for helping me and guiding me through this study project. With his
help, I was able to find my area of interest in microfluidics.
I could not have understood the concepts well enough without the help of the MMNE Lab and the Lab
Technicians. Seeing the multitude of applications of droplet-based microfluidics was a huge help in
my research.
I thank Pavar Sai Kumar Sir and Abhishek Kumar Sir for guiding me through each lab session and for
all the help with my project.

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 Birla Institute of Technology and Science-Pilani,
Hyderabad Campus

Certificate

This is to certify that the project report entitled “Applications of Droplet Based Microfluidics”
submitted by Mr. SAMARPITH S ROUT (ID No. 2020AAPS1332H) in partial fulfilment of the
requirements of the course BIO F226, Study Project Course, embodies the work done by him under
my supervision and guidance.

Date: PROF. SANKET GOEL

BITS-Pilani, Hyderabad Campus

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 ABSTRACT

Most MEMS devices consist of the following components:

Actuators – help produce the electrical or mechanical microfluidic forces that circulate the fluid
samples around the chip.
Sensors – measure the electrical, optical, magnetic, or thermal traits of the targeted samples.
Performed by micro-electrodes or optical sensors.
Electronic circuits – amplify the signals or reduce noise to help make the test results clearer.
This report aims to explain the working of two of the most profitable and successful applications of
droplet-based microfluidics – inkjet printing and lateral flow assays (or strip tests).
The emergence of the inkjet printer represents the first multi-billion-dollar success in the
microfluidics industry. Inkjet printers (both household and office printers and commercial high-speed
printers) use Microfluidic droplet technology to print ink on the paper or printing surface.
Microfluidic devices are small plastic chips that contain channels for transporting fluids. These
channels can be as narrow as 75 microns.
Microfluidic devices have both mechanical and economic advantages and hence is considered for
many applications and is a top 10 revolutionary technology candidate.
When fluids flow through these channels in a micro scale, they follow a laminar flow (fluids flow side
by side without mixing) instead of in a turbulent fashion (where inertia causes the fluids to mix and
have a more random motion). This laminar flow introduces a challenge in mixing fluids. Extra
mechanisms must be added to mix fluids via diffusion, which can add to costs and complexity.
However, the path of flow of these fluids is predictable, which is an advantage.
Test Strips or Lateral Flow Assays started in the 1960s with diabetes testing. Then, they became
commercialised capillary driven test strips in the 1980s. These use fleeces to draw fluids up the strip
via capillary effect and produce a qualitative result of whether a sample contains a particular
substance. These tests make it easy for anyone to administer, and the colour coded results are simple
and effective in giving a clear and concise reading to the end user.
The main challenge of lab on a chip remains to be end to end integration – combining all the
processes performed in a laboratory on a single chip, instead of just specific processes.
Organ-on-a-chip has revolutionised clinical trials and development of treatments for various diseases
and ailments. It simulates real conditions inside the human body and more precise as it can be
personalised to any group of people.

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 CONTENTS

Title page…………………………………………………………………………………….1
Acknowledgements ………………………………………………………………………….2
Certificate ……………………………………………………………………………………3
Abstract ………………………………………………………………………………………4
Introduction ………………………………………………………………………………….6
How Microfluidic Devices are Manufactured…….
…………………………………………………………………………..7
Inkjet Printing ……………………………………………………………………………….12
Lateral Flow Assays …………………………………………………………………………16
Organ-on-a-chip ……………………………………………………………………………...20
Conclusion ……………………………………………………………………………………23
References …………………………………………………………………………………....24

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 INTRODUCTION

Photolithography has given rise to the manufacturing methods of microfluidic devices. However,
methods have also evolved since then, resulting in better yield and better quality.
Inkjet printers and Lateral Flow Assays are two of the most successful and most revenue generating
applications of Droplet Based Microfluidics.
Inkjet printers, in a personal use scale, use air pressure differences (back pressure) and the viscosity of
ink to put ink on paper.
On commercial applications, high speed printers are used to print labels and manufacturing and
expiration information on sold items. These printers put out a continuous stream of ink (which flows
in the form of a jet) and uses electric fields to separate this stream into droplets. This is called Digital
microfluidics or dielectrophoresis.
Both applications result in reliable output of drops of ink on a printing surface.
Strip tests, or lateral flow assays, have huge applications in the field of medicine. They are used as
easy-to-administer tests to help diagnose certain diseases or conditions. Their main motive is to be
easily portable and distributable, and easily accessible and usable by the public, as opposed to lab
technicians or medical professionals.
Fleeces are the regions that the strip tests are divided into; each fleece has a particular function.
Lateral flow assays use different types of fleeces – separation fleece to separate different fluids,
reaction fleece that contains suspended chemicals for testing (immobilised antibodies that bind to the
desired object) are used in Lateral Flow Assays.
Strip Tests have been incredibly useful in diabetes and pregnancy test markets. However, there is still
room for improvement – these tests lack pinpoint accuracy, and do not give quantitative results, as
most of the time results are shown as a colour on a strip. Also, mixing involves added mechanisms
and cost because of the laminar flow of fluids in the strip.
Organ-on-a-chip has revolutionised clinical trials and development of treatments for various diseases
and ailments. This comes at a crucial time, after the outbreak of SARS-COV-2 virus. This method has
various benefits over petri-dish cultures and animal testing, as it can be more accurate as it simulates
real conditions inside the human body and more precise as it can be personalised to any group of
people. This makes this method easily usable and extremely crucial for administering treatments for
specific ailments, which may affect different people in different ways.

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 HOW MICROFLUIDIC DEVICES ARE MANUFACTURED

PDMS (polydimethylsiloxane) is the most widely used polymer for manufacture of microfluidic
channels because of its biocompatibility, optical transparency, and non-toxicity.

Soft Lithography
This is done to create a negative master pattern on a silicon wafer substrate.
Steps –
1. Create a hydrophilic substrate surface on Silicon substrate. Oxygen plasma treatment is used
to raise the surface energy of the substrate. This results in the required adhesion between
silicon wafer and photoresist on the final master pattern.

Flat water bubbles demonstrate the treated wafer’s hydrophilicity

2. Spin-coat the Silicon wafer using photoresist. This is done using a spin coater, which ensures
even coating of photoresist on the wafer.

3. Soft Baking the wafer. This evaporates some of the solvent from the photoresist, which
improves adhesion between the photoresist and the substrate, and allows the UV photo mask
to be applied without sticking.

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4. UV exposure to cure the photoresist. The photo mask (designed on CAD software) is placed
on the wafer.

5. Post exposure bake. This promotes mechanical relaxation of the microfluidic channels to
avoid stresses. The pattern should be visible on the wafer after sufficient exposure.

6. Developing the channel. Development timings are dependent on the width and height of the
channels. The wafer is submerged in the developer fluid in a glass dish.

The wafer is then taken out and the photoresist is rinsed off.

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7. Dry the Silicon wafer and allow it to cool to room temperature.

Silicon master wafer ready to create PDMS device

Using the master pattern to create a PDMS microfluidic device

1. PDMS is poured on the wafer and allowed to cure for 3 to 4 hours.

2. Then the PDMS is cut out of the wafer.

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3. Inlet and Outlet holes are punched on the PDMS.

4. Oxygen-plasma bonding is used to seal the cover glass on the PDMS.

5. Epoxy resin is used to seal tubes to inlets and outlets. Leaving the device at 60 degrees
Celsius for about 3 days significantly improves bond strength.

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 Manufacture using the Bioprinting Method
A cast is created in a petri dish and microfluidic channels are printed on it using Pluronic F-127 as the
printing material. A skirt is printed around the microfluidic device to ensure that the PDMS stays in
place.

This must be then casted in PDMS. PDMS is poured evenly over the cast in the petri dish.

It is then cured in the petri dish for about 24 hours in an ambient temperature of 25 degrees Celsius.
Then, the PDMS is peeled off and the Pluronic F-127 is washed off the chip.

Inlet and outlet holes are then made and the chip is attached to a glass slide or plate for stability.

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 INKJET PRINTING

Household/Office Inkjet Printers

Images are produced as a collection of small dots (pixels). These dots are produced by drops of ink
being discretely released from numerous nozzles in a controlled manner.

Small circumference of the nozzle and the back pressure inside it will not allow ink to leak. The width
of the entire chamber and nozzle (the black part of the image shown above) is less than that of a
strand of hair.
When ink needs to be released, the ink in the nozzle is vaporised into a bubble by use of a heating
resistor. The bubble expands and pushes the ink out of the nozzle.
After the primary droplet is released, there is no more current flow through the heating resistor, hence
causing the ink to cool and contract. During this contraction, outside air fills the vacuum created by
the ink retreating into the nozzle. This is where the back pressure of the ink in the nozzle plays an
important role. The bubble collapses in about 20 microseconds.

Surface tension of the ink at the meniscus will cause fresh ink to fill in to the tip of the nozzle,
allowing for another drop to be formed and released. The meniscus settles in about 50 microseconds.

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During formation of the droplets, two droplets are formed – a larger primary droplet and a smaller
satellite droplet. These two droplets combine on the paper to form a drop of ink and settle on the
paper as a single dot. 150 to 300 million of these drops are produced per second.

High Speed Droplet Formation – Continuous Inkjet Printing

Continuous Inkjet Printing involves a continuous stream of ink that exits the nozzle, at which point it
is broken into droplets which results in the droplets of ink forming an image on the printing surface or
substrate. This is used on commercial inkjet printers (CIJ) that are used for printing labels on food
packaging. (expiration dates, batch codes, names, product logos, etc.)
The process begins with a high-pressure pump used to direct liquid from a reservoir to micrometre
sized nozzles.
Advantages of this method of printing are –
 High throughput
 High droplet velocity
 Increased distance from printhead to substrate
 Reduces the chance of nozzle clogging due to continuous operation
Thanks to these positive attributes, this printing mechanism is used for not only printing on paper, but
printing various materials (even live cells) and the manufacture of modern OLED displays.

Droplet formation in a flowing liquid is caused by its tendency to minimize its surface area as it tries
to occupy its most thermodynamically stable state. Hence, for a liquid, that would be a spherical
‘droplet,’ rather than a cylindrical jet.
The surface area to volume ratio of a sphere is double that of an optimised cylinder of the same
radius.

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The stream of liquid must compete against inertia to form such a droplet – as inertia (and Newton’s
First Law) force the flowing liquid to keep doing so in a jet- like shape. The minimization of surface
area causes some liquid to move up (against gravity) to form a droplet.

Droplets in a freely falling liquid are formed on the later end of the stream, and not as soon as the
liquid exits the nozzle.
However, for printing purposes, the droplets must be formed immediately. Hence, the fluid is vibrated
at a frequency roughly equal to the frequency of formation of the droplets.
This is achieved via a piezoelectric crystal that vibrates the nozzle.

As the ink exits the nozzle, there is formation of two types of droplets – the primary droplet and a
smaller satellite droplet that forms immediately after the primary droplet.
These satellite droplets are separated using charge electrodes and high voltage plates that act as
actuators – using electrical energy to control the flow of the liquid and separate its components.

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The larger charged particles deflect more that its smaller counterparts, hence only the primary droplets
land on the printing surface or the substrate while the satellite droplets are collected as unused ink.

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 LATERAL FLOW ASSAY

Lateral flow tests are a diagnostic test that can be delivered at the point of care. Results from these
tests can be obtained quickly, easily, and affordably. These tests are used for detection of Covid -19,
HIV, and Ebola, among others. Lateral Flow Assays can be in a dipstick or cassette format.
These tests will determine if there is a specific antigen present in the patient’s sample of their bodily
fluids (blood, saliva, or urine).
The sample is placed on a well and is transported via channels from the sample pad to the conjugate
pad. These pads are made of specific materials like natural cellulose.

The conjugate pad consists of antibodies attached to nanoparticles. The antigens, if present in the
sample, will bind with the disease specific anti-bodies in the conjugate pad and flow as an
antigen/antibody nanoparticle complex.

These complexes will subsequently bind to more nanoparticles on the test line. These are called test
antibodies, and these will determine the result of the test.

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As more of these combinations happen, a coloured strip will emerge, indicating the result of the test.
The control line will appear coloured regardless of positive or negative test, to show the test’s validity.
The control line consists of capture antibodies, which ensure that the test has been done correctly.

To achieve a coloured result, the conjugate pad antibodies contain a ‘visual particle.’
A wick pad, or flow pad, is used to ensure that the fluid travels effectively from the top to the bottom
of the test using capillary action.

In the above image, the grey element is the flow pad. The yellow arrows indicate direction of flow.

Developing a Lateral Flow Assay

Antibodies are dispensed onto a strip of nitrocellulose.

Capture Antibody is dispensed on the strip.

The strip is dried in an oven for 30 minutes.

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The conjugate antibodies (with a labelled component that causes colour change in the test) is allowed
to dry down and dispense on the conjugate pad.
Then, the detection label is dispensed. The reagent is not immobilized, but is stored at the required
position, so that it is allowed to dry down. This enables the Lateral Flow Assay test to be performed in
various rugged conditions that may not be otherwise conducive in a laboratory. This is a huge
advantage of these tests, as they allow administration at the point of care (at a less equipped medical
centre or even at home).
After the detector label is dispensed, it is allowed to dry on a screen for one hour.

Finally, the dried dispensary agents, nitrocellulose membrane with immobilised capture antibody, the
conjugate pad sprayed with detector antibody, and the wick pad is combined.

This strip (shown above) is cut into various final strips, which are then placed in protective plastic
cassettes, and hence, the assay takes the form of the recognisable strip test.

How at-home Covid Tests Work

The sample is put into a buffer solution, which, in this case, is 99.7% salt water.
The buffer solution reagents also consist of a conjugate acid- base pair. This is formed by a conjugate
acid (sodium hydrogen phosphate) and a conjugate base (potassium dihydrogen phosphate). These
two components ensure that a constant pH of 7.4 is maintained. This is the pH level of our blood;
thus, this conjugate acid-base pair helps mimic the conditions in our body.

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This test reagent is then put onto the sample well of the test, which consists of gold nanoparticles. The
nanoparticles act as the visual indicator. These will scatter light and result in the red colour that
appears on the control and/or test lines.

The sample and the gold nanoparticles travel along the conjugate pad, where Covid-19 antigen
particles are present. If the sample consists of Covid-19 antibodies (i.e., if the sample is positive for
Covid-19), these antibodies will bind to the antigens in the conjugate pad. These get stuck on the ‘T’
line, and results in a colour change.

The control line consists of Anti-chicken IgY antibodies, which ensure the validity of the test.

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 ORGAN-ON-A-CHIP

Traditional methods for development of drugs to treat ailments is not the most successful or efficient.
Traditional methods involve testing on human cells on a petri dish, or animal testing, both of which do
not produce desired results a lot of the time.
Human cells need an environment like that of the human body to function and respond to drugs in the
desired manner.
Organs-on-a-chip consist of living cells in a dynamic environment interacting with different cell
types. They are made of bio-compatible material that is modular in nature. This modularity allows
sensors and actuators to be connected to the chip, allowing for analysis or manipulation of the cells.
Several different microfluidic channels serve different purposes of fluid transport, cleaning cells,
discarding waste, and electrically and mechanically manipulating the system as required.

These aim to create the smallest functional unit that represents the biochemistry, the function, and the
mechanical strain that cells experience in our bodies.

Lung-on-a-chip

In an organ-on-a-chip designed to mimic respiration in the body, there are 3 main channels present-
the middle channel consists of lung cells and capillary blood cells separated by a membrane, which is
sandwiched between two vacuum channels with the ability to shrink and expand, which simulates the
mechanical forces that these cells go through when we breathe. These channels are made of PDMS
(polydimethylsiloxane). PDMS is the most widely used polymer for manufacture of microfluidic
channels because of its biocompatibility, optical transparency, and non-toxicity.

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The upper channel (blue), as shown above, is an air channel. A liquid containing nutrients is passed
through the channel below (red), hence called the blood channel. In more sophisticated systems, the
chip can include multiple layers of cells to make a more comprehensive model and to study cell
interactions.

The vacuum channels shown above are in their compressed state, which allows more air to pass
through the air channel, much like inhalation in our bodies.
Infection-causing microbes can be introduced into the air channel, and human white blood cells into
the blood channel. The white blood cells will permeate through the membrane and attack the
infection, much like what happens in our bodies.

The image above shows white blood cells that have made their way through the permeable membrane
and attacking the infectious germ in the air channel.
Hence, in a lung – on – a – chip, this process can be observed and monitored in real time.

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Similarly, a gut-on-a-chip simulates intestinal activity, where fluids in the channels are under constant
peristaltic motion.

Many functions observed in a human intestine can be mimicked on this chip. Thus, we can simulate
diseases like irritable bowel syndrome, and hence find ways to treat it better.
As the processes that take place in various organs can now be simulated, and all these organ-on-a-
chips have fluids passing through their channels, these chips can be integrated (via common vascular
channels, for example) to form a human-on-a-chip.

Organ-on-a-chips can revolutionise the way clinical trials are performed. These chips can be
‘personalised’ to mimic precisely the conditions in the bodies of children versus that of adults,
conditions among different ethnic groups and among people from different parts of the world, etc.
Hence, while administering medicines, we can do so knowing how the drugs will react to different
people specifically. This decreases the margin for error and unpredictability when it comes to testing
drugs.

Why are these chips made so small?

An obvious reason is for better portability and ease of use. Also, smaller channels allow for easier and
quicker transport of fluids through the microfluidic channels. However, additionally, it has been
shown that nanopatterning the surface of the channels in an organ-on-a-chip can help the cell in
growth, differentiation, and adhesion, as well as boost protein expression levels.

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 CONCLUSION

Hence, droplet-based microfluidics have applications that have been tried, tested, and used
successfully for decades.
However, there are improvements that need to be made in these fields.
The main challenge of strip tests or lateral flow assays is end-to-end integration. This involves getting
all processes in a laboratory done on a single chip, instead of specific processes only.
Strip tests are essential to public welfare and efficient diagnosis, and hence efficient treatment.
However, these need to be made available for a wider range of diseases and conditions, while
increasing accuracy and efficiency.
Over 4 crore people around the world are HIV positive. 90% of these people have never been tested
for the virus before. This is because patients are unable to receive exact counts of CD4 lymphocyte in
the blood to track the infections progression. These counts are found using flow cytometry.
This requires a full-scale lab and technicians – it would be better if MEMS devices could make having
this test on a single chip of silicon possible.
Prepared sample is passed through the silicon chip via channels, where different actions like mixing,
separating, filtering are performed.
These actions lead to a result that is shown to the end user. However, the challenge remains – figuring
out how to get the results without requiring technical expertise or intervention by the end user.
Organs-on-chips will lead us to have a much better success in finding cures and treatments, and at a
much faster rate, which can be life-saving for the entire global population. These can also be used for
preventative treatments. For instance, an individual’s stem cells can be studied on such a chip to find
out how they respond to treatments or different conditions as compared to someone else’s stem cells.

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 REFERENCES

https://www.youtube.com/watch?v=NAESOEjcYfc&t=806s - Microfluidics and the Elusive Lab-on-

a-Chip
https://www.youtube.com/watch?v=0PKFQciUWBU - Inkjet Printers | The interesting engineering
behind them
https://www.google.com/url?sa=i&url=https%3A%2F%2Fresources.duralabel.com%2Farticles
%2Fthermal-printers-vs-inkjet-printers-which-printer-should-you-
choose&psig=AOvVaw0ykpp23rdLnKlxay96r4df&ust=1698555831991000&source=images&cd=vfe
&opi=89978449&ved=0CBQQjhxqFwoTCMjJpdr7l4IDFQAAAAAdAAAAABAE (working of
inkjet printers)
https://www.youtube.com/watch?v=-DckWNwE7R4 - The Engineering of Droplets and their
Formation in a Commercial Inkjet Printer
https://www.youtube.com/watch?v=S15tgvIbpBI - Continuous Inkjet Printing - CFD Software for
Microfluidics
https://www.flow3d.com/continuous-inkjet-printing/ - Continuous Inkjet Printing
https://www.youtube.com/watch?v=9yeZSaigBj4 - How inkjet printer work
https://www.youtube.com/watch?v=z07CK-4JoFo - See how a lateral flow immunoassay works
https://www.youtube.com/watch?v=pNRiLh-u9cc - How does a lateral flow test work?
https://www.youtube.com/watch?v=o4iJgz9ugy4 - The Fundamentals of Lateral Flow Devices
https://www.youtube.com/watch?v=vmdvmz64v34 - Timeline for Developing a Lateral Flow Assay
https://www.youtube.com/watch?v=2B-iZGNiPA0 - Chemist Breaks Down How At-Home Covid
Tests Work | WIRED
https://www.youtube.com/watch?v=5xZC8GJs-i4 - COVID 19 Antigen Rapid Test - Animated Video
https://www.youtube.com/watch?v=CpkXmtJOH84 - Geraldine Hamilton: Body parts on a chip
https://www.youtube.com/watch?v=whjBR9CafXI – Organs – on – a – chips
https://www.youtube.com/watch?v=dxz10In1774 - Bioprinting 101: How to make Microfluidic Chips
https://www.youtube.com/watch?v=lH-FCSxRvrU - How to make a microfluidic device

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