design and development of a multiport microfluidic device
TRANSCRIPT
Design and Development of a Multiport Microfluidic Device for
In-Vitro Surface Stimulation of the Retina
BY
ASHWIN RAGHUNATHAN
B.E., Rajalakshmi Engineering College, Chennai, India, 2012
THESIS
Submitted in partial fulfillment of the requirements for the degree of Master of Science in Mechanical Engineering
in the Graduate College of the University of Illinois at Chicago, Illinois, 2016
Chicago, Illinois
Defense Committee: Laxman Saggere, Chair and Advisor Thomas J. Royston, Bioengineering John B. Troy, Northwestern University
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This thesis is dedicated to my parents and my grandparents, who have been beside me and motivating
me throughout my life.
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Acknowledgement
I would like to thank my advisor Prof. Laxman Saggere for his guidance, support and patience in
accomplishing this thesis. His expertise and vision in MEMS has been an inspiration through all stages of
my thesis. He has always directed me the right direction and motivated me to work harder and I will
always be thankful for providing me with this opportunity. I am grateful to Prof. Thomas Royston and Prof.
John B Troy for providing their precious time and serving on my thesis committee. This research has been
supported by NSF grant 0938072. I am also grateful to NCF staff, Dr. Seyoung An and Dr. Khodr Maamari,
for their guidance and training with microfabrication processes.
I would like to thank my labmates Ishan Shinde, Dr. Corey Rountree, Dr. Thomas Lucas, Anirudh
Katti and Pradeep Kumar for their constant support and cheering. I would like to thank Corey for carrying
out the rat experiments, helping with setting up experiments and sharing his knowledge and expertise.
Finally, I would like to express my profound gratitude to my parents for providing me with
unconditional support and affection throughout my years of education and my research. This
accomplishment would not have been possible without them
Ashwin Raghunathan Chicago, IL May 13th 2016
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TABLE OF CONTENTS
CHAPTER PAGE
1. INTRODUCTION………………………………………………………………………………….....................................1
1.1 Background and motivation……………………………………………………………………………………............1
1.2 Need for a device……………………………………………………………………………………………………………….3
1.3 Thesis objective and specific aims………………………………………………………………………………………4
1.4 Literature review………………………………………………………………………………………………………………..4
1.4.1 Epidermal Drug Delivery Devices………………………………………………………………………………………..5
1.4.2 Implantable Drug Delivery………………………………………………………………………………………………….5
1.5 Thesis organization…………………………………………………………………………………………………………….6
2. DESIGN AND MODELLING…………………………………………………………………………………………………………..7
2.1 Functional and geometric requirements for device design…………………………………………………7
2.2 Device design specifications…………………………………………………………………………………………….10
2.3 Device Design………………………………………………………………………………………………………………….10
2.3.1 Design of Microchannel and Ports on Silicon Layer………………………………………………………….11
2.3.2 Design of Reservoir on Glass Layer………………………………………………………………………………….12
2.4 Complete Device Structure………………………………………………………………………………………………13
2.5 Device Modelling……………………………………………………………………………………………………………..16
2.5.1 Simulation and validation of fluid flow through single port micropipette………………………..16
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2.5.2 Simulation of device microchannel………………………………………………………………………………….19
3. DEVICE FABRICATION AND BONDING………………………………………………………………………………………21
3.1 Silicon layer fabrication……………………………………………………………………………………………………21
3.1.1 Process flow…………………………………………………………………………………………………………………….21
3.1.2 Mask design…………………………………………………………………………………………………………………….23
3.1.3 Device fabrication process………………………………………………………………………………………………24
3.1.3 Visual characterization of fabricated silicon layer……………………………………………………………26
3.2 Fabrication of glass layer…………………………………………………………………………………………………28
3.3 Bonding of the layers and tubing interface……………………………………………………………………..29
3.3.1 Anodic bonding of the layers…………………………………………………………………………………………..29
3.3.2 Tubing Interface………………………………………………………………………………………………………………31
4. EXPERIMENTAL CHARACTERIZATION AND TESTING OF THE DEVICE………………………………………33
4.1 Experimental characterization and results……………………………………………………………………….33
4.2 Validation of experimental data with modelling………………………………………………………………36
4.3 Interfacing IAMM device with retina……………………………………………………………………………….38
5. CLOSURE…………………………………………………………………………………………………………………………………..40
References………………………………………………………………………………………………………………………………………..41
APPENDIX………………………………………………………………………………………………………………………………………….44
VITA………………………………………………………………………………………………………………………………………………….45
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LIST OF TABLES
Table I: Measured dimensions of ports under microscope. ....................................................................... 28
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LIST OF FIGURES
FIGURE PAGE
1. Retina cross section……………………………………………………………………………………………………………………2
2 Experimental setup and geometry (a) Schematic of the experimental setup. (b) pMEA used for
in-vitro experiments. (c) Close-up image of the electrodes in an array…………………………………….8
3 Schematic of a 3 x 3multiport device overlaid on 8 x 8 electrode array, T-shaped pattern formed
by actuation of selective ports…………………………………………………………………………………………………..9
4 Conceptual 3D model of the IAMM device………………………………………………………………………………..11
5 Design of silicon layer (a) CAD drawing of the silicon layer (Top View), (b) 3D model of the silicon
layer and close up of the array of ports, (c) Isometric section view A-A’……………………………………12
6 Design of glass layer (a) 2D CAD drawing (b) 3D model of glasslayer………………………………………..13
7 Sectional view of 3D model (a) Mid-section isometric view (b) alignment of glass and silicon layer
at the inlet………………………………………………………………………………………………………………………………..14
8 3D rendering of IAMM device……………………………………………………………………………………………………15
9 Dimensions of micropipette (left) and solid model (right)………………………………………………………..16
10 Validation of experimental and simulation data of volume injected for micropipette at pressures
(a) 0.3 psi, (b) 0.5 psi, (c) 0.8 psi, and (d) 1 (psi)………………………………………………………………………….18
11 (a) Solid model of microchannel fluid component. (b) Direction of flow in a microchannel……….19
12 Simulated flow of microchannel into retina (a) Flow simulation into the retina. (b) Increase in
spatial spread with increasing pressure…………………………………………………………………………………….20
13 Simulated data of microchannel interfaced with retina…………………………………………………………….20
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14 Microfabrication process flow for silicon layer………………………………………………………………………….22
15 Three masks for photolithography: (a) Mask 1: Mask with alignment marks for BSA, (b) Mask2:
Mask with channels, and (c) Mask 3: Mask for multiport with close-up image of the ports on the
mask…………………………………………………………………………………………………………………..…………………….23
16 Alignment marks used for alignment of microchannel and microports (a) alignment mark on
mask 1, (b) alignment mark on masks with channel and ports, (c) Alignment marks formed after
alignment of both masks and exposure…………………………………………………………………………………….24
17 Fabricated silicon layer and zoomed in 2D image of etched channels and ports under electron
microscope……………………………………………………………………………………………………………………………….26
18 Microscope images under 10x 0.3 objective (a) Microchannels (b) Microports…………………………27
19 Silicon layer dimensions under microscope, top (left) bottom (right)………………………………………..27
20 Fabricated borosilicate glass layer…………………………………………………………………………………………….28
21 Anodic bonding. (a) Anodic bonded IAMM device held in a forceps. (b) Anodic bonding setup. (c)
Schematic of anodic bonding setup…………………………………………………………………………………………..29
22 Tubes and connectors used to connect the device with pressure injector. (a) Schematic of tubes
interfaced with the IAMM device. (b) Bonded IAMM device interfaced with tubes and
manipulation rod. (C) Union and luer connection linking interface between IAMM device and
pressure injector…………………………….………………………………………………………………………………………..31
23 Experimental characterization of the device. (a) Experimental schematic for characterization. (b)
Experimental setup for characterizatio………………………………………………………………………………..……33
24 Change in height of the meniscus after cycles of injection………………………………………………………..34
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25 Volumes of liquid ejected from the IAMM device at various input pressures characterized using
the meniscus tracking method.………………………………………………………………….................……………..35
26 Microchannel and micro port interfaced with water domain in COMSOL model…..………………….36
27 Simulation for pressures 0.3, 0.6, 0.9 psi…………………………………………………………………………………..36
28 Model correlation of experimental and simulation for 30ms…………….………………………………………37
29 Model correlation of experimental and simulation data for 50ms………………….…………………………37
30 Experimental set-up for device interfacing with the retina. (a) Picture showing the IAMM device
over the MEA before contacting the retina (b) Picture showing the details of tubing connectors
between the pressure injector and the IAMM device.……………………………………………………………...38
31 IAMM device lowered down to interface with retina (a) Device before coming in contact (b) Device
in contact with retina…………………………………………………………………………………………………………………….39
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SUMMARY
Millions of people are affected by photoreceptor degeneration diseases, which cause permanent
vision loss, and currently there is no cure available. Retinal prostheses using electrical current have
emerged as a promising approach to treat and partially restore the vision lost to these diseases. However,
electrical based prostheses have limitations in terms of providing high-resolution naturalistic vision. As an
effective alternative approach, researchers are exploring stimulation of the retina with neurotransmitter
chemicals. The feasibility of this approach was recently demonstrated by injecting glutamate chemical
into the rat retinas using single port micropipettes. To advance this approach further, there is a need to
inject nanoliters of chemicals at multiple points simultaneously over a 2D surface of the retina in order to
study patterned stimulations through in-vitro experiments. Motivated by this need, this thesis was
undertaken with the objective of developing a specialized multiport microfluidic device with individually
addressable microports and tiny on-chip reservoirs.
A multiport microfluidic device was designed to satisfy a number of functional requirements for
in-vitro chemical stimulation experiments, allowing for injection of chemicals on the top side of the retina
while recording the neural signals with a multielectrode array on the bottom side of the retina. The device
was designed to feature a 3x3 array of 10 µm diameter outlet ports on the bottom side, with each port
independently connected to a tiny on-chip reservoir storing the chemical on the top side via a
microchannel, and a means for external pneumatic actuation for ejecting the stored chemicals through
the outlet ports. The overall footprint area and thickness of the device were 1 sq. cm and 1.3 mm,
respectively. A finite element model of the device was created and its output flow characteristics for
various input pressures were analyzed. A prototype of the device was then built in two layers: a bottom
silicon layer and a top glass layer. The bottom silicon layer containing the delivery ports and microchannels
was microfabricated using conventional micromachining techniques, while the top glass layer containing
the reservoirs was fabricated using ultrasonic machining process. Both layers were anodically bonded to
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SUMMARY (CONTINUED)
create a complete prototype of the device. The eight reservoirs in the top layer were filled with glutamate
chemical and each reservoir was independently coupled to an 8-channel pneumatic pressure injector via
tubes and unions, and the device was experimentally characterized for volume injected at the outlet ports
for various input pressures between 0.1-1 psi, and validated by the finite element simulation results.
Following the characterization, the device was interfaced with explanted retinas, and by actuating the
delivery ports selectively, the chemical was injected into retina through different combinations of ports
and corresponding patterned 2D chemical stimulations of the retina were successfully demonstrated.
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1. INTRODUCTION
Millions of people are affected by diseases such as age-related macular degeneration and retinitis
pigmentosa, which cause photoreceptor degeneration in the retina and ultimately cause visual
impairment [1, 2]. Currently there is no cure available to restore the visual functionality lost caused by the
diseases. Retinal prosthesis is a promising approach as a treatment where retinal neurons are stimulated
with artificial stimulation. Over the decades, there have been significant advancements in developing
retinal prosthesis to stimulate the retina with electrical pulses [3]. A few research groups are exploring an
alternate approach, with the chemicals are delivered to artificially stimulate the retina, proving
advantageous in some aspects compared to electrical approach [4]. The feasibility of the chemical
approach has been demonstrated using a single port micropipette [5]. To advance further, a device for
delivery of chemicals at multiple site of the retina through multiple ports is proposed. The need for such
a device, background, motivation and objectives are detailed in following sections. This thesis discusses
the design, development, and testing of novel MEMS based multi-port microfluidic device for delivery of
chemicals into retina.
1.1 Background and motivation.
The retina is a thin and light-sensitive tissue present in the posterior of the eye, which is
responsible for processing visual signals. The Fig. 1 shows the general scheme of neural circuitry in a retina,
which comprises of multiple layers of specialized cells, interconnected in synapses, each performing
different functions. The rods and cones together form the photoreceptors (PR), which is on the posterior
side and retinal ganglion cells (RGC’s) are in the anterior side of the retina.
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Figure 1: Retina cross section [6].
Photoreceptors are the primary light sensitive neurons in the tissue. The light passing through the
pupil is absorbed by photoreceptor cells and causes the release of neurotransmitters, which trigger chain
of events through the cells in the retina. The signals are directed through the optic nerve to the brain
where visual signals are processed [7]. Diseases such as age-related macular degeneration and retinitis
pigmentosa cause photoreceptor degeneration [1]. Since photoreceptors are an integral part of the retina,
photoreceptor degeneration causes irreversible blindness in patients.
As part of the treatment approaches, retinal prosthesis have emerged as a promising method for
restoring vision by artificial stimulation of retina. Many research groups have been working for decades
on retinal prostheses that stimulate retinal cells by passing electrical pulses using an array of electrodes
[3]. Retinal prostheses based on electrical stimulation are being developed as a restorative aid, but current
electrical-based prosthesis technology is limited in its ability to provide high-resolution natural vision [4].
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To overcome these limitations, a microfluidic-approach is proposed where the retina is biomimetically
stimulated with native neurotransmitters. The advantages include higher resolution and cell specific
stimulation [5]. The feasibility of a chemical-based retinal stimulation has been demonstrated by injecting
glutamate (primary neurotransmitter) into the retina from the PR side or RGC’s side using a single port
micropipette [5]. To advance the chemical-based retinal stimulation further, the stimulation of retina at
multiple sites simultaneously is to be investigated. The stimulation at multi-sites is essential to enable 2D
stimulation over the surface of retina. Injection of small volumes from subset of ports selectively could
possibly stimulate retina to form patterns.
1.2 Need for a device.
To achieve 2D surface stimulation, there is a need for a delivery device comprising an array of
delivery ports that can be individually addressed and for interfacing with the retina for in-vitro studies.
This device is required to have several special design features to enable patterned stimulation of the
retina. These features include:
An array of ports; each port approximately 10 µm in diameter, separated by 100-200 µm.
Ability to withstand pressure up to 10 psi.
On-chip reservoir to store glutamate on order of 500 µL.
Ability to interface the outlets with the retina and external pressure injector with the inlet side.
Each outlet port to be individually addressed by a pressure injector inlet.
Footprint is to be small enough to fit in a petri dish.
The above-mentioned special features require consideration of geometric, fabrication and functional
constraints. To meet theses design requirement, MEMS-based design and fabrication approach is
essential.
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1.3 Thesis objective and specific aims.
As discussed in previous section, the broad objective is to design, fabricate and test an individually
addressable multiport microfluidic (IAMM) device, which is to be interfaced with an explanted retina for
in-vitro studies. Four specific aims of this thesis are:
1. To design an IAMM device considering all functional requirements and constraints.
2. To fabricate an IAMM device.
3. Characterize the performance of the IAMM device.
4. To test the IAMM device functionality by interfacing with retina for patterned stimulation.
1.4 Literature review.
This section details the literature survey of the MEMS-based drug delivery devices. MEMS
technology has wide applications for e.g. lab-on-chip, biosensors, drug delivery in field of medicine and
biology. Based on the broad objectives and aims, this review is limited to research on drug delivery
devices.
Conventionally, drugs have been delivered through mouth, skin, and injection, but recent
advances in MEMS technology have helped to build devices to deliver drugs directly at the tissue level [8].
MEMS and microfluidic technology have helped deliver nano/pico liters at microscopic level, either on
body or inside the body as an implant. The MEMS based drug delivery systems are engineered to permit
fluid flow at micro scale with devices based on microneedles, micro pumps, and micro reservoir [9]. MEMS
technology has been explored for drug delivery but there has been limited research for ocular drug
delivery [10]. MEMS based epidermal drug delivery devices and implantable drug delivery devices are
briefly reviewed.
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1.4.1 Epidermal drug delivery devices.
Epidermal drug delivery devices are commonly available device, mostly skin patches with a drug
reservoir that deliver controlled volumes over a period. The most common form available uses an array
of microneedles that pierce through the dermis for injection. These patches can be used for instant drug
delivery or for over period to deliver drugs like insulin for diabetic patients [11, 12].
Microneedles are one of the most common approaches for drug delivery with many different
variations like solid, hollow, coated and dissolving. An array of these microneedles can be used as
transdermal patch to bypass the epidermis and deliver necessary chemicals and compounds to particular
cells or tissues. However, a major issue conflicting with research requirements is the passive actuation of
microneedle patches, which necessarily result in long drug release times [13-15]. Research groups have
been able to combine PZT micro pumps with silicon microneedles to achieve controlled active transdermal
drug delivery with single or multiple ports [16, 17].
1.4.2 Implantable drug delivery.
Depending on the application, implantable devices are custom designed and fabricated. When
compared with epidermal devices, there are more factors involved in designing implantable devices and
each device must be specially optimized for each application with regards to factors like biocompatibility,
device life, relative size and reservoir capacity [18, 19]. In past few decades, many groups have
investigated implantable drug delivery devices and have made great progress. For example, an
implantable MEMS micro pump has been demonstrated by delivering controlled volumes of cancer
treatment drugs in rat. This device has a micro reservoir with a single port outlet, made of glass and
parylene [20].
Very few research groups have developed MEMS based devices have been developed for
applications to the eye. Li et al., designed and fabricated a parylene based intraocular drug delivery device
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that delivers a drug through a check valve actuated by an electrolytic pumping mechanism, permitting the
delivery of nL/min over long periods while maintaining refill ability [21]. Noolandi et al. have proposed
used an inkjet printer head, comprised of 20 µm diameter outlets that are each 200 µm apart and filled
with neurotransmitters to stimulate PC12 cells in petri dish [22]. The same group also designed and
fabricated a chip where fluid flow was controlled by electro-osmosis. The device has four openings of 5
µm aperture, each connected to separate channels [23].
Tu et al. have investigated chemical retinal prostheses using similar methodologies to design and
fabricate a parylene-based multiport device with 8x8 outlet microneedles, with an intended purpose of
chemically stimulate the retina [24]. However, the device performance was demonstrated by piercing
through chicken tissue, but not interfaced with a retina.
1.5 Thesis organization.
As outlined in objective, the scope of the thesis includes design, development and testing of a
novel IMM are discussed in detail in subsequent chapters. Chapter 2 details the design for the IAMM
device based on requirements and constraints, and fluid flow simulation of the IAMM device to determine
the volume injected for variable pressures. Chapter 3 describes the process flow to fabricate the designed
prototype and interfacing the IAMM device with tubes for pneumatic actuation. Chapter 4 describes the
experimental characterization of IAMM device performance and simulation of the fluid flow. Chapter 5
discusses the testing of the IAMM device interfaced with the retina for patterned stimulation by
selectively actuating subset of delivery ports. The conclusion, discussion of the entire work and future
work possible is presented in chapter 6.
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2. DESIGN AND MODELLING
A need for a specialized device for multi-site stimulation of the retina was described in chapter 1.
This chapter presents detailed geometric and functional requirements for the IAMM device. The design is
obtained considering the geometric, fabrication and functional constraints. The IAMM device comprises
of multiple layers, the design of each of the layers is described. Entire IAMM device was modeled and
simulated to determine the performance and was simulated in COMSOL, a Multiphysics simulation
software.
2.1 Functional and geometric requirements for device design.
The ultimate objective of the IAMM device is to study in-vitro stimulation of the retina. The IAMM
device has to satisfy many constraints of the experimental setup. These constraints include both the
chemical delivery system and the system to record the responses of the retina in-vitro. To understand all
the constraints, it is important to consider the experimental setup in which the device will be used for
chemical stimulation of the retina in-vitro while simultaneously recording the corresponding responses.
The details of the experimental setup are shown in Fig. 2(a) and constraints are discussed below. Some of
the main components of the experiment are multi-electrode array (MEA), pneumatic pressure injector, 3-
axis manipulator and perfusion setup. During the experiment IAMM device is required to be positioned
over retina for manipulation so that ports are above the retina and the chemicals stored in the device is
to be ejected through the ports using external pneumatic actuation. The IAMM device is to fit within the
perfusion chamber and maneuvered within the perfusion chamber.
The physiological responses of the retina is captured using a multi-electrode array (MEA), the MEA
that is considered is a perforated MEA (pMEA) (60pMEA200/30iR-Ti, Multichannel Systems MCS GmBH).
The pMEA has 64 electrodes in grid of 8 X 8 and each of a diameter 30 µm and distance in between is 200
µm (Fig. 2(b)). The perfusion chamber has a diameter of 25 mm and 8 mm tall. During the experiment,
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the retina is placed on the pMEA with RGC’s side down and perfused with oxygenated media to keep it
alive. The delivery ports IMM devices must be interfaced with the retina and the inlet of the IAMM device
is to be connected to the pressure injector, which has an inlet opening of 1 mm in diameter as shown in
Fig. 2(a).
Figure 2: Experimental setup and geometry. (a) Schematic of the experimental setup. (b) pMEA used for
in-vitro experiments. (c) Close-up image of the electrodes in an array.
The separation of the electrodes on the pMEA is 200 µm. Fig. 2(C) shows the layout of the
electrodes of pMEA interspersed between perforations. Important functional consideration for IMM
devices is that the chemical delivered through the multi-ports have to form grid pattern and to stimulate
the cells around the electrode. Therefore, the ports are to align with the electrodes when in contact with
the retina as in Fig. 2(a). For simplicity, the preliminary prototype is considered to have ports in an array
of 3 x 3, of which 8 are active ports and space between the ports is 200 µm, the same as in the MEA for
the convenience of the spatial referencing between ejection point and the cells response. Another
consideration of functional requirement was that the chemical stored in the device be ejected through
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subset of ports selectively to form patterns. An example of such pattern stimulation is by selectively
actuating delivery of chemicals through subset of ports forming a “T” shaped pattern as in Fig. 3.
Figure 3: Schematic of a 3 x 3multiport device overlaid on 8 x 8 electrode array, T-shaped pattern formed
by actuation of selective ports.
To accomplish patterned ejections of chemicals through IAMM device over the duration of the
experiment, the IAMM device is required to hold sufficient amount of glutamate to last through several
cycles of ejections. To enable patterned stimulation through ports, each port must have independent
reservoir that is actuated independently. Several cycles injections are performed into the retina during
the duration of the in-vitro experiment. Based on the amount of chemical injected in each cycle and
duration of a typical stimulation cycle i.e. a few minutes of injection time, it is estimated the individual
reservoir connected to each port should store about 500 µL glutamate.
As the device is lowered over the retina during interfacing, it is essential to determine the moment
when the devices is in contact with the retina. This contact is determined with principle of resistance
change with Ag-AgCl electrode in a conducting solution. To enable the implementation of Ag-AgCl wire
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for contact detection purposes, the IAMM device must provide means to pass the Ag-AgCl electrode and
store the conducting solution.
2.2 Device design specifications.
As highlighted in previous section the IAMM device has to meet several constraints. Based on the
constraints and requirements the design specifications for the IAMM device are detailed below:
1. The entire device footprint is 1 cm x 1 cm and approximately 1 mm tall, considering the
need for the device to manipulate inside the chamber and to move over the electrodes.
2. These delivery ports are to be 10 µm in diameter and in an array of 3 x 3.
3. Multiport outlets are to be spaced at 200 µm to record cell responses at multiple
electrodes.
4. A separate channel for each of the ports linking ports and the pneumatic pressure source.
5. To move the device above the electrodes and be manipulated with 3-axis
micromanipulator.
6. Small tiny hole is required through the center of the device to thread an electrically
conductive wire into an Ag-AgCl solution to determine contact interface of device with
retina.
7. The device should be able to withheld pressures up to 10 psi and inject nanoliter volumes.
8. An on-chip reservoir for each port to hold approximately 500 µL of glutamate for the
entire duration of the experiment.
2.3 Device design.
To satisfy design specifications, a conceptual design was established. The device comprises of two
layers where the top layer would comprise of reservoirs, bottom layer would comprise of ports and
microchannel (Fig. 4). The top layer (borosilicate glass layer) and bottom layer (silicon layer) are of
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different material and are to be bonded anodically to form an integrated device. Based on the constraints
a conceptual model is designed and shown in Fig. 4.
Figure 4: Conceptual 3D model of the IAMM device.
2.3.1 Design of microchannel and ports on silicon layer.
The fabrication techniques and its feasibility were considered before designing the channels and
ports on silicon wafer. For initial prototype, only nine ports were considered designed in an array of 3x3.
Considering the constraints of microfabrication, ports were designed on one side of the silicon wafer and
channels on the other side of the wafer. The port depth was definite to be 100 µm and rest of the thickness
of the wafer was for microchannel as shown in the isometric sectional view (Fig. 5(c)). The thickness of
the wafer used was 340 µm as shown in Fig. 5(c).
The ports are designed in an array of 3 x 3, spaced 200 µm apart that can be aligned above pMEA
and designed at the center of 1 cm x 1 cm die. Each port is 10 µm in diameter. To address the channels
separately, channels are designed in a circular patter as seen in Fig. 5(a). Fig. 5(a) below shows the CAD
drawing of the channels aligned with the ports.
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Figure 5: Design of silicon layer. (a) CAD drawing of the silicon layer (Top View), (b) 3D model of the
silicon layer and close up of the array of ports, (c) Isometric section view A-A’.
Microchannels are arranged in a circle at a radius of 3800 µm from the center and 8 ports have
individual channels of their own. The larger end of the channel is 1200 µm and the narrow end has a width
of 120 µm. The central port of 50 µm at the center aligns right above the center port in 3 x 3 array Fig.
5(a). The need for the central port and dimensioning of the channels is explained in section 2.4. The
enlarged image shows the outlet ports, which are thru holes. Fig. 5(b) shows 3D visual of the proposed
silicon layer with multiple channels and ports.
2.3.2 Design of reservoir on glass layer.
Top layer helps sealing the channels and acts as a reservoir for the IAMM device. Openings for
pneumatic pressure input are designed above the larger end of the microchannel. Hence, the holes for
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these openings are laid out in a circular pattern with their centers on a circle of radius 3200 µm as shown
in Fig. 6(a). Tubes connected from the pressure injector are to connect through the glass layer to create a
closed system. There are many commercially available tubing, but based on industry standards and easy
availability, 1.58 mm outer diameter (OD) tube was considered. The openings on glass layer are 1.6 mm
in diameter. Fig. 7 shows 2D sketch of the top layer with dimensions considered. The central hole on the
glass layer is 250 µm (Fig. 6(b)) also aligns with the center port on the silicon chip.
Figure 6: Design of glass layer. (a) 2D CAD drawing (b) 3D model of glass layer.
2.4 Complete device structure.
Although the designs of both layers are expounded separately, entire device is designed as a whole.
Certain design feature can be explained combining both layers together. Mid-section isometric view of
the 3D model is shown in Fig. 7, where the alignment of the glass layer and silicon layer is displayed. Top
layer has opening of 1600 µm and larger end of the microchannel below is 1200 µm, this dimensioning
forms a step, which is shown in Fig. 7(b). The step formation seen in Fig. 7(b) helps the tube to be placed
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right on top of silicon layer, which aids in air to flow through channel without any blockage through the
channel and the port.
Figure 7: Sectional view of 3D model (a) Mid-section isometric view (b) alignment of glass and silicon layer at the inlet.
The silicon layer has alignment marks on sides of the channel at distance 100 µm from the channel
edge, which as seen in Fig. 5(a) are used to align the circumference of the hole on glass layer with silicon
layer for anodic bonding. Aligning the inlets on glass layer to these marks eases placement of the layers
together for bonding.
As seen in Fig. 5(a) and Fig. 6(b), both layers have a central hole of 50 µm and 250 µm, which can
be used for threading the electrode used to determine the contact with retina. The holes are aligned in
the center from 250 µm on glass layer, 50 µm on top side of silicon layer and connecting with 10 µm port
on the bottom. These holes are to be filled with AgCl solution and threaded with electrode. Fig. 8 is a 3D
rendering of IAMM device held in an electrode holder and positioned inside perfusion chamber of an
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MEA. The top view shows the relative size with respect to chamber and edges to allow perfusion flow in
the chamber.
Figure 8: 3D rendering of IAMM device.
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2.5 Device modelling.
In this section, the designed IAMM device is modelled and simulated to determine the volume
dispensed for variable pressure input interfaced with the retina. Modelling a retina accurately is beyond
the scope of this thesis, hence to determine the resistance of the flow, the retina is bulk modelled. The
actual properties of the retina are unknown. Thus, the volume injected of the single port micropipette
interfaced in a retina is simulated to determine the resistance to the flow. These validated boundary
conditions are defined to simulate resistance to the flow for the IAMM device interfaced with the retina
and determine the volume injected in retina. The volume injected of the micropipette was acquired based
on an experimental characterization with single port interfaced with the retina [5]. Glutamate was
injected into the retina and the volume was characterized using meniscus tracking method. Data
assimilated was for pressure ranging between 0.1-1 psi and for injection times of 10-100 ms. Under similar
boundary conditions, the pipette is substituted with device model and expected volume injected can be
simulated.
2.5.1 Simulation and validation of fluid flow through single port micropipette.
The experimental volumes injected were acquired under experimental conditions, which is to be
replicated as boundary condition for the model. The injections were performed for pressure ranges of 0.1-
1 psi, injection pulse duration of 10-100 ms and at frequency of 3-5 Hz. Dimensions of the pipette
measured under microscope as in Fig. 9 (left).
Figure 9: Dimensions of micropipette (left) and solid model (right).
17
The dimensions of the micropipette are measured accurately under the microscope (Fig. 12 left).
The micropipette is modelled to the same dimensions with the tip interfaced with the surface of the
modeled retina.
Retina is permeable and highly porous material with multiple layers of cells. Each retina has a
different structure within, considering its non-uniformity the retina is bulk modelled for accelerated
simulation. The modeled domains are imported to COMSOL and form an assembly with identity pairs
between domains. Material is an important factor to determine the flow is into a porous media. Pipette
is modelled as a fluid volume of water. Retina is a tissue present on the posterior part of the eye. Most of
the body tissue is made of water, density and dynamic viscosity defined for retina is similar to water [25].
Properties like porosity and permeability provide membrane like structure to the model.
Pressure is varying parameter, which is defined globally. The simulation is run to replicate the
experimental setup with same parameters as mentioned in previous chapter. Hence, a time dependent
laminar study is selected for the same, which uses the Navier-Stokes equation for conservation of
momentum and continuity equation for conservation of mass. The pressure applied for the experiments
are pulsed at frequency of 2 Hz and pulse time of 30 ms, 50 ms. The material defined is separate for both
domains. The pipette being modelled as a fluid volume was defined as water. The retina is bulk modeled
as a porous material, the properties like porosity and permeability defines the solid model as a tissue. The
study is time dependent over a period of 2 seconds and simulated for multiple pressures defined using
parametric sweep. The sweep ranges from 0.1-1 psi with each injection for 30 ms. The time set for the
solver is from 0-2 s with step of 0.01 s.
The data obtained from the experiment was for volume injected for the applied pressure. The
data available is for pressures 0.3, 0.5, 0.8, 1 psi. The data is available for injection time of 10-100 ms.
18
Figure 10: Validation of experimental and simulation data of volume injected for micropipette at pressures (a) 0.3 psi, (b) 0.5 psi, (c) 0.8 psi, and (d) 1 psi.
The above plots (Fig. 10) are for injection time vs volume injected/injection for different
pressures. The experimental data images acquired are analyzed manually, could be subjected to some
human error. In addition, the retina is a complicated structure, which is made of multiple layers. For the
above simulation, retina is assumed porous uniformly and is bulk modelled. Hence, considering the above-
mentioned possibility of error and assumption, the data compiled is not accurate but gives a range of
volume injected to be expected.
Based on the plots above, it was concluded the boundary layer applied for the model is similar to
the actual experimental setup. Under similar conditions, the micropipette can be replaced with the
designed IAMM device. The simulated data from the device provides the range of volume injected
expected from the actual IMM device.
19
2.5.2 Simulation of device microchannel.
As discussed earlier, the volume injected can be determined under similar boundary conditions
to simulate the resistance to flow. For efficient simulation, only the fluid volume inside the channel in
modelled and defined as the fluid component. Due to the symmetry of the design, only one microchannel
is pathway is considered. The Fig. 11(a) shows the modelled single micro channel.
Figure 11: (a) Solid model of microchannel fluid component. (b) Direction of flow in a microchannel.
The retina from the previous model is modelled at the surface of the outlet of the microchannel.
The fluid flow is shown in Fig. 11(b). The microchannel is simulated under same exact boundary condition
as the micropipette to simulate the volume dispensed to be expected.
This simulation provides the plots for volume injected and Fig. 12(a) shows the flow from the
port outlet into the retina. The average velocity is determined at the outlet of these ports to determine
the average volume injected/second.
20
Figure 12: Simulated flow of microchannel into retina (a) Flow simulation into the retina. (b) Increase in spatial spread with increasing pressure.
The simulation also shows different pressures affecting the flow spatially as in Fig. 12(b). The
following is the plot (Fig. 13) for 10 µm diameter port, for volume injected at different injection time for
pressures from 0.1-1psi.
Figure 13: Simulated data of microchannel interfaced with retina
0
1
2
3
4
5
6
7
8
10 20 30 40 50 60 70 80 90 100Vo
lum
e in
ject
ed (
nL/
inje
ctio
n)
injection time (ms)
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
21
3. DEVICE FABRICATION AND BONDING.
In this chapter, the microfabrication process steps for the designed IAMM device are detailed.
Section 3.1 presents the process flow for the silicon layer and microfabrication process steps based on the
sequence. Glass layer fabrication is discussed in section 3.2. Further, both the layers are bonded anodically
to form a device and interfaced with the pressure injector using tubes and unions, all of which is presented
in section 3.3.
3.1 Silicon layer fabrication.
A process flow for silicon layer was defined based on the design required. Conventional
microfabrication processes are used fabricate the silicon layer. The process flow, mask layout used and
microfabrication processes are discussed in subsequent sections.
3.1.1 Process flow.
Process sequence is created considering on the design requirement and conventional
micromachining techniques. The silicon layer was fabricated with microfabrication processes and the glass
layer was fabricated by conventional ultrasonic machining (Bullen Ultrasonics, Eaton, Ohio).
Micro fabrication processes are in 2D, as per design the dimensions of channels and ports are
different hence they have to be aligned and etched separately from top and bottom side of the wafer
which necessitates use of double side polished silicon wafer. Considering the aspect ratio of the channels
and ports, deep reactive ion etching is the only process for deep channel etching. This process requires a
designed masking layer; Aluminum is deposited as masking layer. Multiple masks are used for patterning
the designed channels and ports on the photoresist using photolithography. The microchannels are
exposed from the top side; the ports are aligned from the bottom side and exposed. Based on the
22
fabrication processes needed the process flow for silicon layer is show in Fig. 14. The process flow
constitutes of eight steps with two photolithography steps with 3 masks.
Wafer Cross-section Process Description
Silicon wafer, double side polished 4in and 340 µm thick.
Deposition of 400 nm of aluminum, e-beam vapor deposition.
Spinning of photoresist on both sides.
Patterning the channels on top side.
Development of the pattern in developer 400k, ration 1:4 with DI water.
Patterning Backside with ports using BSA (Backside alignment).
Using PAN etch (Aluminum etch), to form the masking layer and stripping the photoresist.
Performing plasma etch with DRIE and etching the microchannel for 240 µm.
Etching the backside with DRIE for 100 µm.
Dicing wafer using dicing saw.
Stripping of the aluminum masking layer with PAN etch.
Anodic bonding between glass and silicon layer
to make a device.
Figure 14: Microfabrication process flow for silicon layer.
(a)
(b)
(c)
(d)
(e)
(i)
(j)
(f)
(g)
(h)
23
3.1.2 Mask design.
The IAMM device design was patterned on the silicon wafer for fabrication. Three masks were
designed based on the fabrication process flow and shown in Fig. 15.
Figure 15: Three masks for photolithography: (a) Mask 1: Mask with alignment marks for BSA, (b)
Mask2: Mask with channels, and (c) Mask 3: Mask for multiport with close-up image of the ports on the
mask.
The mask is 4 inch x 4 inch for a 4-inch wafer, with 24 devices and symmetric as a design. The Fig.
16 shows the sequence shows the alignment marks used in the masks and after exposure alignment
marks.
24
Figure 16: Alignment marks used for alignment of microchannel and microports. (a) alignment mark on
mask 1, (b) alignment mark on masks with channel and ports, and (c) Alignment marks formed after
alignment of both masks and exposure.
These are main alignment marks used to align the other two masks. The three alignment marks
are of dimensions 150 µm, 75 µm and 10 µm from largest to smallest. Theses marks are placed in a straight
line passing through the center of the mask as in Fig. 15 (a). The micro channels are designed on mask 2
shown in Fig. 15(b) where each device is designed inside a 1 cm x 1 cm boundary of thickness 1 mm.
Boundary designed isolates each device and aids in easier dicing.
As seen in Fig. 15(b), six dies are designed in each quadrant and have space of 2.2 mm in between.
The devices on the edges have a single alignment mark next to them, which was used to confirm the
alignment. A mask 3 as in Fig. 15(c) has multiple ports and is in the same layout as the microchannel.
3.1.3 Device fabrication process.
The fabrication processes are executed in a sequence determined in the process flow.
Accordingly, the first step is deposition of masking layer for DRIE process. Masking material are decided
based on number of etch cycles, etch time. For deep trench etching using DRIE, aluminum has a high
selectivity of 105 as masking material and etch rate as small as 10-3 nm/min [27]. A 15 nm layer of
chromium is deposited to promote adhesion. The aluminum layer is deposited for thickness of 450 nm
using e beam vapor deposition at rate of 250 A0/min. The rotary motion of wafer holder within the
chamber is set at 25 rpm to provide uniform deposition. Deposition is followed by photolithography to
25
pattern the design on the wafer and form an aluminum-making layer. One side of the wafer was spun with
photoresist AZ1518 at 1500 rpm for 40 seconds and prebaked at 100 0C for one minute. Mask 1 with
alignments marks is exposed on the photoresist using Karl Suss MA6 Aligner. Post exposure the wafer is
developed in a 400K AZ developer for 30 sec. The same surface is exposed with Mask 2 and aligned as
seen in Fig 14. The exposure wavelength is 400 nm and exposed for 12 s. The wafer is hard baked for two
mins at 110 0C to increase the strength of patterned photoresist. Other side of wafer is spun with AZ 1518
and prebaked at 100 0C for a min. For the third exposure, the alignment mark developed on channel side
is aligned with marks on mask 3 and then exposed on other side of the wafer. The ports are patterned on
the bottom side of the wafer and hard baked for 2 mins.
Exposed aluminum region formed is etched using PAN etch and the thin layer of chrome beneath
is etched immersing in chromium etchant forming masking layer needed for DRIE of silicon. DRIE provides
the anisotropic etching needed to fabricate vertical smooth walls. Top surface with microchannel is etched
with recipe used for 1066 cycles to depth of 240 µm. Aspect ratio is to be considered for etching of small
features to higher depth. Aspect ratio is ratio of width to depth. For microchannel, the aspect ratio is 1:2
which is achievable. The ratio for the ports is higher, which calls for a different recipe. In case of higher
aspect ratio the etch rate is smaller and deposition cycle is longer. To achieve smoother etch for small
features chamber pressure for both cycles is increased, duration for deposition cycle is increased to 6
seconds and gas flowrate to 80 sccm. Ports are etched through at rate of 1 µm/min for 650 cycles. Optical
profilers with UV light source cannot detect the surface at such narrow and deep trenches. The device
was visually seen under dark field microscopy and electron microscope. Fig. 17 below shows 2D image of
the etched device and the zoomed-in image under electron microscope after etching.
26
Figure 17: Fabricated silicon layer and zoomed in 2D image of etched channels and ports under electron
microscope.
Next step is cleaning the wafer and dicing each device using a dicing saw of width 200 µm. Dicing
saw is programmed to cut across the wafer at specific distances to isolate each device. As the design
layout was symmetrical, saw is programmed to cut through specific points in X and Y-axis. Each device is
isolated and aluminum making layer is stripped using PAN etch and cleaned.
3.1.3 Visual characterization of fabricated silicon layer.
After fabrication, the silicon layer was visually inspected under an optical microscope to
determine the quality of DRIE etch and measure port size. The fabricated silicon layer was inspected under
Nikon ECLIPSE Ti-E, an inverted microscope system. The microscope is connected to an ANDOR Zyla 5.5
sCMOS camera to capture the image seen under the microscope. At 5.5 megapixel, the camera can
capture an image resolution of 2048 x 2048. Nikon imaging software, NIS-Elements, was used to control
the microscope settings to display accurate images and capture quality images. The Fig. 18 is captured
under the microscope with an objective Plan Fluor 10x 0.3 under bright field.
27
Figure 18: Microscope images under 10x 0.3 objective (a) Microchannels (b) Microports.
The Fig. 19 shows the image of the array of ports imaged using objective Plan Fluor ELWD 20x
0.45. The light source from the top side of the microscope passes through the open ports. The lens is
focused on the surface from top and bottom side of the silicon layer. The images were captured and the
port diameter annotated using NIS-Elements software (Fig. 19).
Figure 19: Silicon layer dimensions under microscope, top (left) bottom (right).
On inspection, it was seen that some of the ports were not open completely. As shown in Fig. 19,
the image was take from the microchannel side and from the port side and measured. Port 2-8 are open
but port 1 is closed. Intended dimensions of the ports was 10 µm, however resultant dimensions were in
between 25-30 µm on average. These errors could be attributed to uneven distribution of plasma, further
28
leading to uneven etching. Top and bottom ends of ports are measured under inverted microscopy as
seen table I.
Port Number Radius on microchannel
side (µm) Radius on port side (µm)
2 8.98 12.6
3 10.49 10.65
4 10.85 12.69
5 7.32 10.54
6 13 13.14
7 10.33 11.8
8 12 12.12
Table I: Radii of the ports fabricated in the silicon layer as measured under an optical microscope.
3.2 Fabrication of glass layer.
The design of the glass layer is shown in Fig 6 in section 2.3. The top layer is made of borosilicate
glass and fabricated by conventional ultrasonic machining. The glass layer was fabricated by an outside
company (Bullen Ultrasonics, Eaton, OH) according to the specifications described in section 2.3.2. To
accomplish this a special tool was made to machine the glass layer. The glass layer (Fig. 20) was fabricated
by direct or indirect use of abrasive material by means of ultrasonic vibrating tool.
Figure 20: Fabricated borosilicate glass layer.
29
3.3 Bonding of the layers and tubing interface.
After the fabrication and dicing of the silicon wafer, the silicon die is anodically bonded with glass
layer. The bonded IAMM device is then interfaced with the tubes connecting the device to the pressure
injector. The IAMM device is attached to a 3-axis manipulator with certain hypodermic steel tubes.
3.3.1 Anodic bonding of the layers.
The silicon and glass layer are aligned and anodically bonded to form an integrated device. Fig.
21(c) shows a schematic of the process done for the device.
Figure 21: Anodic bonding. (a) Anodic bonded IAMM device held in a forceps. (b) Anodic bonding setup.
(c) Schematic of anodic bonding setup.
Silicon end is connected to the positive terminal and acts as an anode and the conducting metal
on the top layer acts as the cathode and connected to a high voltage supply (Stanford Research Systems,
PS325). Both the surfaces are cleaned prior to the bonding, as small particles or air can lead to voids in
the bond causing leaks. The silicon and glass pieces are placed on the metal plate and aligned under
30
microscope. The alignment mark on the silicon layer aligns with a portion of circumference of the hole in
the glass layer. All the alignment marks are aligned and a metal plate is moved to the top of the glass
layer.
This plate is maneuvered using 3-axis manipulator and placed on the glass as in Fig. 28(b), which
completes the circuit. This whole setup is on top of a hot plate (Thermo Scientific, SP131325) with
temperature display. Temperature is set at 475 0C and the applied voltage is 1100 V. The complete
bonding of the device takes approximately 15-20 minutes. This bonding is irreversible and device is sealed
and leak-proof as in Fig. 21(a). The reservoir aligns above the channel and isolates each channel and can
be addressed individually.
31
3.3.2 Tubing interface.
To actuate to fluid in the reservoir, the IAMM device is connected to a pressure injector (PM8000
Programmable Multichannel Pressure Injector, MicroData Instruments, Inc.).The Fig. 22(a) shows the
schematic of complete tubing.
Figure 22: Tubes and connectors used to connect the device with pressure injector. (a) Schematic of tubes
interfaced with the IAMM device. (b) Bonded IAMM device interfaced with tubes and manipulation rod.
(C) Union and luer connection linking interface between IAMM device and pressure injector.
A stiff hypodermic metal tube of OD 1.6 mm is perpendicular to the center of the IAMM device.
This steel tube telescoped high tolerance plastic tube around it, with an inner diameter of 1.6 mm as seen
in Fig. 22(b). This provides better grip over the IAMM device covering more space on the glass layer. This
manipulation rod is glued to the top surface of the glass by JB Weld, which is waterproof and provides a
32
strong adhesion. This manipulation rod is held tight in an electrode holder of OD 1.6 mm, which is
attached to the 3- axis manipulator.
IAMM device is interfaced with the pressure injector using Dupont FPA tubes, PEEK ferrule and
female luer. The tubes with ID 1 mm and OD 1.58 mm is placed in the holes on glass layer and as seen in
Fig. 22(b) the tube sits on the top surface of the silicon. For the same purpose, holes on glass layer was
designed larger than the larger end of the channel on silicon layer as discussed in chapter 2. The pressure
injector has standard tubes connected with female luer at the ends. The tubes connected to the IAMM
device is fitted with 1/16 conical ferrule at the open end as in Fig. 22(c). A female-male luer union is used
to connect the tubes from the IAMM device and the pressure injector. This PEEK union is threaded tightly
with the ferrules preventing any air leaks. Each of the 8 inlet of the reservoir on the device are connected
to each of the 8-channel on the pressure injector, hence each port can be actuated separately.
33
4. EXPERIMENTAL CHARACTERIZATION AND TESTING OF THE DEVICE.
The fabricated IAMM device was characterized to measure the volume injected at different
pressures before using it for in-vitro retinal experiments. The meniscus tracking method is a technique
used to characterize the volume injected of the IAMM, where the meniscus is easily visualized through a
microscope. The trials are run for different injection times and limited cycle. The characterized data was
also validated with a simulation under boundary conditions similar to characterization experiment.
4.1 Experimental characterization and results.
The IAMM device was characterized to determine the volume injected through the ports for
different parameters like pressure, time of injection and duration of all injections. Suction is used to fill
the device through the ports into the microchannel with the device immersed in the liquid. The ID of this
tube is 1 mm and liquid forms a concave meniscus in the tube. Experiment setup for characterization is
seen in Fig. 23(b).
Figure 23: Experimental characterization of the device (a) Experimental schematic for characterization. (b) Experimental setup for characterization.
34
For the IAMM device application of in-vitro experiments, the ports had to be immersed in the
perfusion media. Hence, to mimic similar conditions the ports were characterized immersed in a petri dish
filled with water as seen in Fig. 23(a), which is a liquid-to-liquid flow.
For the setup (Fig. 23(a)), IAMM device manipulation rod is held inside an electrode holder and
moved using a manipulator. Images from the microscope were captured with Moticam 5.0, which is
connected to the computer and triggered using MATLAB. The DAQ captures the initial image of the
meniscus with the camera. After the last injection, DAQ again captures an image. Change in meniscus
height is visible between two images and be measured (Fig. 24).
Figure 24: Change in height of the meniscus after cycles of injection.
The pressures applied were 0.3 psi, 0.6 psi, 0.9 psi and each of this pressure was applied for
duration of injections of 30 ms, 50 ms. Frequency of injection was 2 Hz and number of injections was 25.
Each cycle had 25 injections and was repeated 5 times for all the available ports. Each image acquired for
the above setup is recorded directly to the computer. All the images from 5 sets of injections recorded for
each pressure and injection time are stacked together and visualized in Image-J software. As seen in Fig.
25, the meniscus is tracked after each set of image. The difference in height recorded, is used calculate
35
the volume injected through the ports. The tubes used are cylindrical in shape and assumed as a cylinder.
The difference in the height of liquid before injection (H1) and height of liquid after injection (H2) is the
height of the cylinder. The radius of the cylinder is the ID of the tube used, 1 mm. The volume is calculated
using the equation below:
𝑉 = π𝑟2(𝐻1 − 𝐻2)
where, V= Volume of the liquid injected.
r= radius of inner diameter of tube (mm).
H1= Height of the meniscus before injection (mm).
H2=Height of meniscus after injection (mm).
For each injection time and pressure, the data is averaged for all available ports to obtain one
data point and graph (Fig. 25) shown below is average volume injected for all the ports for the respective
pressure and injection time.
Figure 25: Volumes of liquid ejected from the IAMM device at various input pressures characterized using
the meniscus tracking method.
0
5
10
15
20
25
0.3 0.6 0.9
Vo
lum
e in
ject
ed (
nL/
inje
ctio
n)
Pressure (Psi)
30ms experimental
50ms experimental
36
4.2 Validation of experimental data with modelling.
The experimental data acquired from each port is averaged together. This data was validated with
a COMSOL model run under exact boundary conditions as the experiment for each port. The modelling of
the device is similar to fluid flow model discussed in chapter 2. The same model is used and the retina
domain is replaced with water. The model used to validate the experimental setup is seen in Fig. 26(a).
Figure 26: Microchannel and micro port interfaced with water domain in COMSOL model.
The model was simulated under same conditions as the experiment with a time dependent solver.
Results simulated are discussed below.
Figure 27: Simulation for pressures 0.3, 0.6, 0.9 psi.
37
The Fig. 26 (b) shows the stream flow of the fluid flow and direction. As the simulation is run for
multiple pressure, the Fig. 36 shows the width of the flow which varies with increase in pressure. The plots
shown in Figs. 28 and 29 below compare the volumes of liquid ejected in the experiment and simulation
for injection times of 30 ms and 50 ms.
Figure 28: Model correlation of experimental and simulation data for 30 ms.
Figure 29: Model correlation of experimental and simulation data for 50 ms.
0
2
4
6
8
10
12
0.3 0.6 0.9
Vo
lum
e
Pressure
injection time= 30ms
30ms exp 30ms sim
0
5
10
15
20
0.3 0.6 0.9
Vo
lum
e in
ject
ed (
nL/
inje
ctio
n)
Axis Title
injection time= 50ms
50ms exp
50ms sim
38
The simulation data for each port is averaged similar to see in experimental data plot. Based on
the plots acquired, it can be concluded that the experimental data is in range with the simulation data for
the same condition.
4.3 Interfacing IAMM device with retina.
Following the fabrication and characterizing the IAMM device, it was setup for in-vitro retinal
experiments. The retina was explanted from euthanized wild-type rat and placed over the pMEA and
perfused. The rat dissection and retina stimulation recordings were carried out by Dr. Corey Rountree,
Postdoctoral Research Associate in the department of Mechanical and Industrial Engineering at UIC. All
animal handling and euthanasia were conducted within the guidelines outlined by the National Research
Council’s ‘Guide for the Care and Use of Laboratory Animals’, and all protocols were approved by the
Institutional Animal Care and Use Committee of the University of Illinois at Chicago. The IAMM device was
tested for its performance when interfaced with the retina. The Figs. 30(a) and 30(b) shows the device
setup for the experiment and is discussed below.
Figure 30: Experimental set-up for device interfacing with the retina. (a) Picture showing the IAMM
device over the MEA before contacting the retina (b) Picture showing the details of tubing connectors
between the pressure injector and the IAMM device.
39
The manipulation rod on the IAMM device holds the IAMM device to manipulate over the
electrodes. The manipulation rod was threaded into an electrode holder providing a strong hold over the
IAMM device. This electrode holder was fixed to the manipulator aiding to maneuver the IAMM device.
The 3-axis manipulator holds the electrode holder perpendicular to the setup. The tubing from the IAMM
device is extended until the back of the manipulator and the connectors helps to form a link between
IAMM device and pressure injector. A close up image of the setup is seen in Fig. 30(b) with the device
above the pMEA and perfusion chamber.
Before the experiment, the working of the IAMM device is tested connecting tube to available 8-
channels of pressure injector. The IAMM device was filled up with glutamate using suction, which flows
through the ports and rising in the tubes. The IAMM device is referenced to move the ports right above
the electrodes and using the manipulator. The entire setup is positioned on top of an inverted microscope,
which helps to confirm the contact and alignment of ports with electrodes. The Fig. 31(b) shows the IAMM
device interfaced with the retina.
Figure 31: IAMM device lowered down to interface with retina (a) Device before coming in contact (b)
Device in contact with retina.
The IAMM device was interfaced with the retina and glutamate was injected into the retina. The
stimulation of the retina was achieved.
40
5. CLOSURE
In this this thesis, an individually addressable multi-port microfluidic (IAMM) device is designed
and developed for in-vitro retinal stimulation. The IAMM device was designed and developed to meet
requirements to achieve the objectives specified. An IAMM device was designed considering all
experimental requirements; geometric, fabrication and functional constraints. The designed device was
successfully fabricated using conventional micromachining processes and anodic bonding. The IAMM
device was characterized experimentally to measure the volume injected at the delivery ports and the
experimental data was validated with a fluid flow simulation. The IAMM setup up for in-vitro retinal
experiments and stimulation of the retina was achieved. The thesis objectives described in Section 1.3
were successfully accomplished.
41
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APPENDIX
A. Microfabrication issues and trials.
The recipe for DRIE of the silicon layer was obtained by multiple trials to get the desired result. The
silicon layer with aluminum masking layer was etched for 3 recipes to achieve high etch rate with quality
etch results. Scalloping, a common issue causing uneven formation of sidewalls, was reduced by reducing
the ICP power [28, 29]. Trial recipes were made based on required etch rate around 2 µm/min and smooth
vertical sidewalls. Following table shows the recipes for trials.
Trial 1 Trial 2 Trial 3
Deposition Etch Deposition Etch Deposition Etch
SF6(Sccm) 0 100 10 100 10 100
C4F8(Sccm) 80 10 90 10 75 10
Chamber Pressure (mTorr)
26 30 26
30 23 30
Forward power 28 45 32 45 23 42
ICP (W) 650 800 750 900 650 700
Time(sec) 5 9 6 9 5 9
Table I: DRIE recipe trials for silicon layer.
Silicon was etched for 100 µm for each trial and wafer was probed using Tencor contact profiler.
Passivation gas flow rate is high for each cycle in trial 2, this gave a etch rate as small as 0.8 µm/min. In
trial 1, deposition layer is smaller which gave an etch rate of 1.4 µm/min but high chamber pressure in
etch cycle resulted in small lateral etch causing uneven sidewalls. An etch rate of 1.7 µm was seen for trial
3 with deposition flow rate at 75 sccm and reduced chamber pressure of 23 mTorr during deposition cycle.
Scalloping of sidewalls reduced with decrease in ICP power from 800 to 700 W. Based on the trials, the
silicon layer was etched using recipe for trial 3 with an etch rate of 1.7 µm.
45
VITA
NAME: Ashwin Raghunathan
EDUCATION: B.E. Mechanical Engineering, Rajalakshmi Engineering College, Chennai, India, 2012.
M.S. Mechanical Engineering, University of Illinois at Chicago, Chicago, Illinois, 2016.
POSITIONS HELD: Research Assistant, Microsystems and Devices Laboratory, University of Illinois at Chicago, Chicago, Illinois, 2014-2016.