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Materials Selection and Processing for Reliable
Neural Interfaces
by
Christina M. Tringides
Submitted to the
Department of Materials Science and Engineering
in Partial Fulfillment of the Requirements for the Degree of
Bachelor of Science
at the
Massachusetts Institute of Technology
June 2015
2015 Christina M. Tringides
All rights reserved.
The author hereby grants MIT permission to reproduce and to distribute publicly paper and
electronic copies of this thesis document in whole or in part in any medium known or hereafter
created.
Signature of Author .........................................
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Signature
,,...
.V. .--.-.
..
W...V.
Department of Materials Science and Engineering
May 1, 2015
Signature redacted
. . ....................
Polina Anikeeva
Science
and Engineering
Assistant Prof sor in Materials
i gnnThesis Supervisor
C ertified by............................................................................
Signature redacted
...........
S. Beach
tepartment of Materials Science and Engineering
Undergraduate Committee Chairman
A ccepted by.........................................
1/Geoffre
I
Materials Selection and Processing for Reliable
Neural Interfaces
by
Christina M. Tringides
Submitted to the Department of Materials Science and Engineering on May 1,
2015, in Partial Fulfillment of the Requirements for the Degree of Bachelor of
Science
Abstract
The understanding of the brain would be revolutionized by a tool that can measure
intra- and extra-cellular electrical potentials on a parallelized scale, without
disrupting the neural physiology. Existing technologies do not sufficiently carry
out these functions. Using a thermal drawing process (TDP), multimaterial fibers
comprised of polymer-metal composites can be fabricated to create flexible,
microelectrode arrays. These fibers can be further processed after the TDP, using
selective etching to reduce the diameter of the probe. These devices have been
implanted and have been used to record neural activity in vivo while evoking
minimal tissue response. Additionally, electrodeposition of biocompatible metals
onto the fiber-electrode tips can be implemented to increase the signal-to-noise
ratio (SNR). Here, I describe the electroplating of gold onto the fiber-tips of tin and
tin-indium electrodes, which were drawn using TDP. By adjusting the
electrodeposition conditions, the electrode tip geometries can be tuned to optimize
the interface between the device tips and neuronal membranes.
Thesis Supervisor: Polina 0. Anikeeva
Title: AMAX Assistant Professor in Materials Science and Engineering
2
Acknowledgements
First and most importantly, I would like to thank Professor Polina Anikeeva. She not only
allowed me to be one of the first students in her group, but she spent time in the lab with me to
teach me techniques. Her mentorship and insight over the last three years has led me to become
an independent scientist and fall in love with the world of neural engineering.
I would also like to thank Andres Canales, who mentored me in the lab from day 1. He
was, and still is, always available to answer questions and offer feedback about my experiments,
results, posters, and talks. I am also grateful for his thorough editing of this thesis.
Dr. Ulrich Froriep has helped fuel my excitement for neurobiology and biomedical
related research. His patience, expertise, and ability to answer every possible question about
neuroscience that I can come up with are truly remarkable.
I would like to thank the other members in the Bioelectronics group for their help at
different stages through this work.
Finally, I would like to thank my family for always being so supportive in everything. I
especially want to thank my father, Michael Tringides, who is always available to help me
practice for talks and poster presentations. His willingness to talk through ideas and his many,
many questions has further solidified my own understanding of my work. Of course, I also need
to thank him for taking me to the laboratory when I was 8 years old and going through my first
experiment with me. In addition to seeing an electron beam that day, I saw myself becoming a
scientist, just like you Dad.
3
Table of Contents
1 Introduction
1.1 Existing devices for neural recordings
1.2 Limitations of existing devices
1.3 Thermal drawing process
1.4 Selective etching
1.5 Electroplating
6
7
9
10
13
14
16
2 Materials and Methods
2.1 Materials Selection and Fabrication: Design 1
2.1.1 Materials Selection
2.1.2 Fabrication
2.1.3 Post-TDP Processing
2.2 Materials Selection and Fabrication: Design 2
2.2.1 Materials Selection
2.2.2 Fabrication
2.2.3 Post-TDP Processing
2.3 Selective Etching of Metals
2.4 Characterization
2.4.1 Imaging
2.4.2 Connectorization
2.4.3 Impedance measurement
2.4.4 Soak tests
2.5 Electroplating
3 Results
17
17
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20
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22
22
22
23
23
23
24
25
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28
3.1 Design 1
3.1.1 Cross-section
3.1.2 Flexibility
3.1.3 Impedance
3.2 Chronic Implantation
3.3 Design 2
3.4 Electroplating
3.4.1 Selective Etching of Metal
3.4.2 Characterization
3.4.3 Effects on Impedance
3.4.4 Soak tests
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35
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38
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43
4 Discussion
4.1 Conclusions
4.2 Limitations and Future Work
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44
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51
5 Bibliography
6 Appendix
4
List of Figures
1.1
1.2
1.3
Current neural devices using metal electrodes
Existing devices for recording neural activity with silicon electrodes
The draw tower used in TDP
8
8
12
2.1
2.2
2.3
2.4
2-step TDP
PMMA-COC-SnIn composite preform
Selectively removing metal
Schematic of electroplating
18
22
23
26
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
3.12
3.13
3.14
3.15
3.16
3.17
3.18
3.19
Selective etching of the sacrificial cladding
Etched PPSU-PEI-Sn fiber
Flexibility of the probe
Impedance spectroscopy of Design 1
Neural recording data
Schematic cross-section of PMMA-COC-SnIn fiber
SEM cross-section of PMMA-COC-SnIn fiber
Selective etching of Sn
Gold-capped electrodes
EDs compositional analysis
Porous gold surface
Metallurgical junction
Electroplated gold on SnIn
Impedance spectroscopy of Design 1 with gold plated tips
Impedance spectroscopy for a -500 pm electrode (PEI-Sn fiber)
Impedance spectroscopy for a -300 pm electrode (PMMA-COC-SnIn)
SEM after soak test
Soak tests for a -300 pm electrode in a COC-SnIn fiber
Soak tests, replotted
29
30
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32
34
34
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36
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37
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42
5
1. INTRODUCTION
The brain is composed of numerous cells and of different cell types, which are connected to one
another in intricate ways to give rise to specific functions. To identify exactly which neurons are
involved in particular behaviors, devices which contain electrodes are implanted into the brain.
These electrodes must be able to record neural activity at the single cell level so that the neurons
specific to a particular behavior can be identified. To fully uncover the neural code, these
identified neurons should be stimulated, ideally by functionalities incorporated in the same
implanted device, to see if an observed behavior is induced. Devices with precise recording and
stimulation capabilities would allow for the characterization of the mechanisms of debilitating
neurodegenerative disorders, such as Parkinson's and Alzheimer's disease, as well as greatly aid
the development of therapeutics and treatments for patients.
6
1.1
Existing devices for neural recordings
There are a variety of neural probes used in animals and even in humans. Some electrodes are
simply placed on the outside of the skull, and while these are non-invasive, they have a very poor
signal-to-noise ratio (SNR) and can only provide macroscopic brain activity information.
Devices with electrodes that penetrate into the brain have much better resolution, but have the
risk of causing damage to neural tissues. In these implants, the electrodes can be used to record
or stimulate neural activity. Deep brain stimulation (DBS, Fig. 1.la), amongst the most wellknown of brain implants, uses stimulating electrodes. Multiple large, millimeter-diameter
electrodes are inserted into various regions of the brain during a neurosurgical procedure.
Through these electrodes, voltages are delivered to regions deep in the brain, with pulse
frequency (5-200 Hz) and amplitude (1-10 V) dependent on the particular patient conditions
(Perlmutter and Mink, 2006). DBS requires a very invasive surgery, and while it is helpful for
some patients, there is a high probability that it will lead to psychiatric complications such
obsessive compulsive disorders and depression (Schlaepfer et al., 2014).
Devices that use recording electrodes tend to have less invasive surgery procedures
because the probes are much smaller. This is because to record single-neuron activity, the size of
the electrode must be comparable to that of a neuron (-5-20 pm). Additionally, the electrode
impedance must be high enough to filter biological noise. The interface between the surface of
the electrode and the neuron also influences the quality of signal recorded.
Keeping these considerations in mind, various recording electrodes and electrode arrays
have been fabricated. In these devices, the electrodes are typically based on metals,
semiconductors (silicon) and glasses. While DBS is an example of metal electrodes, there are
also metal microelectrode systems which incorporate more electrodes with a total diameter of
7
hundreds of microns. The electrodes themselves can be bundled together to create multi-array
electrode systems. Examples of this include tetrodes (Fig. 1.1b, Recce and O'Keefe, 1989)) and
microwire arrays (Fig. 1.1c). Metal electrode systems have high variability in the geometry of
the device. Additionally, the scalability of number of electrodes can be limited depending on the
fabrication method.
Fig 1.1: Current neural devices using metal electrodes. (A) DBS electrodes, inserted deep into
the brain of a patient are an example of stimulating electrodes. Two examples of recording
electrodes are the (B) tetrode bundle, www.uq.edu.au, and the (C) Tucker-Davis microwire array
of 16 electrodes.
Fig 1.2: Current devices used for neural recordings, using silicon electrodes. (A)
Utah array, with a 10x10 array of electrodes made of stiff silicon. (B) Silicon
probe with 16 electrodes, from NeuroNexus.
Silicon-based electrodes on the other hand are more reproducible since they are made using
standard microfabrication techniques. Some examples include the Utah array (Fig. 1.2a) and
8
silicon probe arrays (Fig. 1.2b), which have the advantage of giving three-dimensional spatial
recording data. The geometry of the array can be controlled using fabrication techniques
borrowed from the highly developed semiconductor industry. However, these silicon-based
devices are difficult and very expensive to manufacture, and the electrode are typically larger
than those found in metal-based devices. No existing devices allow for high throughput neural
.
recordings in deep-brain structures.
1.2
Limitations of existing devices
Silicon is very stiff and has a Young's modulus that is 130 GPa (Hopcroft et al., 2010), which is
much larger than that of neural tissues, -3 kPa (Soza et al., 2005). The brain is not fixed to the
skull and so it undergoes micromotion during normal subject activity. Implants, however, are
fixed in place (Giletti et al., 2006). As the brain moves, a stiff device would be unable to move
with the organ. An analogy to this is holding a metal spoon in a bowl of pudding, and while
keeping the spoon fixed in place, shaking the pudding. The resulting severe disruption of neural
tissues and breaching of the blood brain barrier (BBB) leads to an inflammatory foreign body
response manifested in an increase of glial scarring (Ward et al., 2009).
This immune reaction is caused during the implantation procedure, and also over time as
the foreign materials remain in the brain. As proteins, most substantially albumin and
immunoglobulins, adsorb to the foreign surface, they trigger the aggregation of glial cells around
the device (Tang and Eaton, 1999). Scar forms between the neuron and electrode, providing an
insulator that decreases the recorded signal and decreased signal-to-noise ratio (SNR) of the
device. Additionally, the density of neurons in the region decreases since cells are being ripped
9
open (Purcell et al., 2009), leading to a significant decrease in the number of neurons in the
region that can be recorded from.
The devices described in Fig. 1.1 and 1.2 are made from materials that have mechanical
modulus mismatch and/or chemical instability with neural tissue. Implants that are small would
be minimally invasive, and limit the initial damage done during implantation. Flexible devices
would be able to follow the movement of the brain and reduce the amount of tissue damage
caused by the micromotion.
Polymers offer a wide range of materials properties including flexibility and various
biocompatibility characteristics, such as surface chemistries that limit protein adsorption or
slightly charged surfaces that electrostatically attract cells (Teramura et al., 2008). The lower
elastic modulus of the polymer, especially when it is thin and less than 1 mm in diameter, would
allow it to move with the brain. A chemically inert system would also reduce the toxicity in the
cells. It would be desirable to combine multiple polymers materials and even metals, to create
multimaterial, multifunctional devices.
This work describes the fabrication of such devices, which are processed by leveraging a
fiber drawing process.
1.3
Thermal drawing process
In order to process multiple materials simultaneously and create a plethora of electrode numbers
and geometries, we employ the thermal drawing process (TDP) that has been used in the
telecommunications industry since before the 1990s to create fiber-optic cables (Goff, Hansen,
1996). While fibers in the telecom industry are typically drawn from glasses, pioneering efforts
from Professor Yoel Fink (MIT) and colleagues have demonstrated the success of using TDP in
10
polymer systems and polymer-metal composites. Diverse materials can be thermally drawn
together provided that the glass transition (T) and/or melting temperatures (Tm) of each material
are comparable so that all the components of the fiber flow similarly to keep their relative aspect
ratios and positions with respect to one another (Bayindir et al, 2005). This means that crystalline
materials should melt below the temperature at which glassy materials flow (viscosity <107
poise) (Abouraddy et al., 2007).
The first step of TDP involves creating a macroscopic template, called a preform, which
contains all the features of the final device. Since the preform has a diameter on the order of
centimeters, it is easy to machine trenches and channels that can be filled with other materials or
even left empty to be used as hollow channels. There are no restrictions on the preform
geometry, only that it must be contained in the furnace (the fiber tower in the Fink lab restricts
preform diameters to <1.5"). Once all of the desired functionalities are incorporated, the preform
is placed in a vacuum oven to consolidate all the materials together. Then, the preform is taken to
the draw tower where it is heated in a three-part furnace, where each the temperature of each
section can be independently adjusted, and tension is applied. As the preform is gradually heated,
because of gravity and the weight attached to the bottom of the template, it begins to flow and
form a neck. Once the fiber is long enough to reach the capstan it has reached the 'bait-off stage,
and the fiber is fed into a capstan. The tension controlled through the capstan speed serves to
stretch or "draw-down" the fiber, such that the cross-section of the fiber is uniform in the longaxis while decreasing the diameter and all the features proportionally.
To change the tension on the preform, the temperatures of the furnace zones can be
adjusted. It is important to control the stress levels in the fiber to ensure the cross-section is both
stable, uniform, and matches that of the preform. The draw-down ratio is determined by the
11
speed at which the capstan spins, and the speed at which the preform is fed into the furnace. By
adjusting these two values, the preform can be reduced in size by up to 200 times. Uncontrolled
stress levels will form a thinner neck and could lead to non-uniform fiber diameters, or even the
breaking of the fiber during the drawing process.
/
C
Fig. 1.3: The draw tower used in TDP (access and training generously provided by Professor
Yoel Fink and his lab group). (A) Image of most of the tower. Shown is an amber polymer rod,
which is the preform holder. The silver cylinder is the three-tiered furnace, and the weight at the
bottom of the image is connected to the end of the preform. (B) Zoomed-in view of the laser
micrometer, where the drawn fiber exits the furnace. (C) Schematic of the preform in the
furnace. (D) Detailed view of the preform necking and leading to the drawn fiber.
12
To further reduce the size of the electrode and to include additional materials and
functionalities, TDP can be repeated multiple times by fabricating new preforms with the new set
of features. This time, however, fibers from previous draws can be consolidated into the new
template and during a consecutive TDP these features will be further reduced in size. Capillary
breakup limits the diameter of the drawn metal electrodes to > 1 pm. Polymers and glasses,
however, can be drawn down to tens of nanometers (Kaufman et al., 2011). A multi-step TDP
can reduce preform features by up to 40,000 times for metals and -1,000,000 for polymers and
glasses. Continuity of electrodes along the fiber is essential for the electrodes to be able to record
neural activity.
1.4
Selective etching
To ensure that the draw process is stable and that the capstan will be able pull the fiber when its
final diameter is reached, the drawn fiber diameter cannot be too small (>150-200 pm for simple
all-polymer structures, and larger more complex polymer-metal composites). We have shown
that with TDP, we can reduce the fiber to diameter to 400 pm. However, if this fiber were to be
implanted, its large size would cause significant glial scarring. To overcome this, each preform is
designed to incorporate an outer sacrificial cladding. The materials of the 'core' and cladding are
chosen to satisfy the TDP temperature restrictions, but they are also selected so that they have
orthogonal solubilities. This means that the cladding is used during TDP only to keep the drawn
fiber diameter large enough for stable drawing conditions. However, once the fiber has been
drawn, solvents can be used to remove just the outside layer, reducing the probe diameter to tens
of microns.
13
In this work, I will describe selective etching used in polymer-metal composites for the
development of implantable neural recording devices. Selective etching can also be used to
remove metal and create micro-wells at the tips of fibers.
1.5
Electroplating
Typically, a useful impedance range to record neural activity is a few hundred kW to a
few MQ. In our previous work, we have found impedances for our tin electrodes ranging from
500 kW to 2 MQ. Electroplating is a powerful technique which has been used to reduce the
impedance of microwire devices, such as tetrodes, so that these electrodes are sensitive enough
to record small amplitude signals (Ferguson et al., 2009). Changing the plating parameters, the
geometry of the tips can be changed to better interact with neurons since the surface area of the
tips can be increased to allow for improved contacts with these cells (Hai et al., 2012).
Though gold is one of the materials most commonly electroplated, work with iridium
oxide (IrOx) indicates that this material yields a higher recording SNR and acts as a more robust
electrode. Using IrOx, the impedance of an electrode could be reduced from 266 kW, when
plated with gold, to 7.1 kW. Additionally, the charge storage capacity of the IrOx plated
microelectrode increased by an order of magnitude (Han et al., 2008) which is of importance for
electrical neural stimulation.
Multiple materials can be used to coat electrode, given that they form stable metallurgical
junctions with one another. Once selective etching removes metal in the fiber and creates
channels, electroplating can be used in a 'layer-by-layer' fashion to first add gold and then
deposit IrOx on top to further tune the electrode surface. Additives, such as carbon nanotubes
(CNTs), can be added to the plating solution and thus will be suspended in the deposited material
14
(Ferguson et al., 2009). This has been show to further reduce tip impedance without increasing
the likelihood of shorting between the neighboring electrodes.
In this work, I will describe how electroplating can be used to reduce the tip impedance
and enhance biocompatibility of the implanted electrodes.
15
2. MATERIALS AND METHODS
As mentioned before, the size of the electrodes must be small to increase resolution of the array
so that only one neuron interfaces with each electrode, and to ensure a high density of electrodes
to record from many neurons. A small size is also important to reduce the biological response
from the implant. Because of this, low-melting temperature metals and soft, biocompatible
polymers are used to create multimaterial composites. The thin diameter of the metals and the
flexible polymers allow the implants that move around with micromotion of the brain. Multiple
polymers can be used to allow for further post-TDP processing. Each metal electrode is
surrounded by a polymer cladding to insulate the electrical signals from one another.
16
2.1
Materials Selection and Fabrication: Design 1
2.1.1. Materials Selection
In addition to the constraints for material selection placed by TDP, the materials should be
chemically stable since they will be in direct contact with neural tissues. The first of two neural
probes systems described in this work contains tin (Sn) metal electrodes (Puratronic@ tin rods,
Alfa Aesar, Ward Hill, MA), which has a Tm of 235*C. This conductive material is surrounded
by a cladding of poly-(etherimide) (PEI, rod, ULTEM 1000, McMaster Carr, Robbinsville, NJ),
with a Tg of 216*C. The final material, used as the sacrificial cladding layer, is poly(phenylsulfone) (PPSU rod, McMaster Carr, Robbinsville NJ) with a T. of 220*C.
2.1.2. Fabrication
The fabrication of this fiber is simplified by using (at least) a two-step drawing process. For the
first preform, a rod of PEI with a diameter of 1.5" was cut to a length of 8". Using a lathe, a
trench was drilled in the center of the rod to fit a tin rod (6 mm diameter, 16 cm long). The tin
was inserted into the core, and then the polymer-metal composite was placed in a furnace for a
month, at 150*C and under vacuum. This process of consolidation is to join all the materials
together to form a single unit.
Once consolidation was complete, the preform was taken to a custom-designed drawing
tower (thanks to Professor Yoel Fink, DMSE, MIT). At the top of the drawing tower is a clamp
to hold the preform in place, and which is used to lower the preform at a controlled speed (the
"downfeed speed") into the furnace. The furnace has three zones whose temperature can be
independently adjusted to control the heating of the preform, and regulate where necking of the
preform takes place. Temperatures in the middle temperature are the highest and are well above
17
the Tg and Tm of the composite components, while the top and bottom temperatures are lowered
so that the preform flow is controlled. Once the drawn fiber exits the furnace, it passes a laser
micrometer which is used to provide information about the diameter of the draw. A tension meter
is positioned below the micrometer to provide real-time on the stress levels in the fiber. The final
component is the capstan which determines the speed at which the fiber is drawn, and can
increased or decreased to increase or decrease the draw-down ratio, respectively.
The preform is mounted first to a preform holder, a simple polymer of rod with machined
grooves to allow wires to connect the preform to the holder. A holder (-500 g) is attached to the
bottom part of the preform, and reduces the time at which bait-off occurs. The holder is marked
at various locations such as 27 cm above each of the following: the approximate desired location
of bait-off, where the Sn metal beings, where the Sn ends, where the preform ends. Since the
furnace is not transparent, these markers allow for a way to have an idea of what part of the
preform is in which zone of the furnace. Since bait-off occurs in the middle zone of the furnace
and the preform is 8" in length, the section of the preform -3 cm below the metal rod was
positioned in the middle of the furnace (and thus is the presumed location of bait-off).
Over the course of 2 hours, the temperatures of the three zones of the furnace were
gradually increased until they reached the bait-off conditions (205 0 C, 355 0 C, 1900 C for the top,
middle and bottom zones, respectively). After -45 minutes, the bait-off location began to soften
substantially and because of gravity, started to move downwards. In less than 10 minutes from
this point, necking occurred and the drawn fiber was connected to the capstan. The temperatures
of the furnace were reduced to 205*C, 325*C and 190 0 C respectively. These temperatures can be
changed during the drawing process to alter the stress levels to ensure a stable draw. The
18
smallest diameter of the fiber was 600 Im. Fig. 2.1 shows the cross-section of the preform and
the final fiber, with a schematic depicting the transition between these states.
For the second preform, a rod of PPSU (1.5" in diameter) was cut to a length of 8", and
again a lathe was used to drill a channel in the center of the rod. This time, a bundle of the
previously drawn PEI-Sn fibers was inserted in the hole. The number of fibers inserted is
dependent on the size of the fibers and the diameter of the lathe drill bit. These fibers can be put
in particular arrangements, and can be further separated from one another by incorporating
pieces of just-PEI fiber. Once the geometry of the array is determined, the preform is placed
under vacuum conditions for 45 minutes at 253 0 C to again consolidate the system. A drawing
process with the same procedures and parameters as the first draw was used, and ultimately a 7electrode array was fabricated. The process described in this paragraph can be further repeated,
this time using a bundle of 7-electrode fibers, to fabricate fibers with an even higher electrode
count.
Fig. 2.1: 2-step TDP. Repeating the process again by fabricating a new preform could further
increase the number of electrodes.
19
2.1.3. Post-TDP processing
At this point, the drawn fiber has a substantial amount of (sacrificial) cladding surrounding it: the
electrode bundle and insulating PEI cladding is only -85 pm, but the fiber has a diameter closer
to 400 pm. Tetrahydrofuran (THF) is an organic solvent which dissolves PPSU at a much
quicker rate than it removes PEI. The fiber can be suspended vertically in THF, and after etching
of the PPSU, the very tip can be cut. Alternatively and for a safer etch, the ends of the drawn
fiber should first be covered with epoxy to protect the cross sections from the solvent and to
ensure only the cladding is removed. The fiber is then placed in a dish and submerged in THF for
25 minutes. To ensure that the PPSU is uniformly removed and doesn't fall to the bottom part of
the fiber, it is best to tape the ends of the fiber to the well, slightly above the solvent level. This
suspension, along with stirring or shaking of the dish every 5 minutes, allows for a complete
removal of the sacrificial cladding. Now, the fiber is just 85 pm in diameter, composed only of
PEI cladding around 7 Sn electrodes.
2.2
Materials Selection and Fabrication: Design 2
2.2.1. Materials Selection
A new, lower-melting temperature composite was drawn. This successful fabrication introduced
PMMA, a transparent and soft polymer, and SnIn into the materials toolbox. The Tg of PMMA is
-105*C which is comparable to the Tg of cyclic olefin copolymer (COC, Boedeker Plastics),
which is 158*C. PMMA is easily removed by acetone, while COC is unaffected by this solvent.
Indium-Tin (In90/Sn1O, Goodfellow) is an alloy with a Tm of -140'C and is selected as the
conductive material.
20
2.2.2. Fabrication
The fabrication of this fiber also requires a two-step drawing process. First, a rod of COC, 1.25"
in diameter and 8" long, is taken to the machine shop and a hole is drilled in the center of the
rod, with a lathe. Then, a 5 mm long rod of SnIn is placed in the core, and the two materials are
consolidated under vacuum. Again, this preform is taken to the drawing tower and the only
difference from the fabrication of design 1 is the temperatures of the furnace chambers. The baitoff temperatures are now 125*C, 265*C and 120*C, in the top, middle and bottom zones
respectively. Once bait-off occurs, the middle zone is reduced to 240*C. The resulting fiber is
approximately 1 mm in diameter.
Then, two slabs of strengthened UV-Resistant PMMA, both 1.5" in diameter, are cut to
be 8" long. Although PMMA rods are not commercially available, it can still be drawn in the
tower. Using an
32
" end mill, 5 channels are created in one of the PMMA slabs which are the
same diameters as the COC-SnIn fibers and which are 6 cm long. The previously drawn fibers
are carefully placed into the trenches, which are deep enough to hold the fibers. Then, the second
piece of PMMA is placed on top, covering the fibers in the channel.
Next, the slabs are taped together at the top and bottom spot of the preform with heatresistive tape. At this point, the preform is placed in a heated press with controlled temperature
and pressure of each plate. Once the preform is placed in the center of the press, the temperature
of each plate is increased to 125*C and the pressure is set to 40 barr. After 1 day (-24 hours) at
these conditions, the preform is removed. Consolidation can be verified by cutting the length of
the preform by a few millimeters and looking at the cross-section of the cut. Uniform polymer
and no indication of a line between the slabs indicate a good consolidation.
21
After this, the new preform (Fig. 2.2) is ready to be drawn. It is placed in the furnace
again and subjected to the same temperature intervals as the first preform. To help all the
components flow more cohesively with one another, the tension is raised by increasing the draw
capstan speed from 0.6 m/min to 1 m/min. Because the PMMA is a rectangular slab, the final
cross-section of the drawn fiber is also rectangular, though the COC-SnIn insulated electrodes
are circular. The final diameter is -520um x -700 pm.
Fig.
2.2:
PMMA-COC-SnIn
composite preform. The COC
insulated SnIn electrodes are
separate from one another by
equal distances. The PMMA will
be removed by acetone and since
COC is unaffected by the solvent,
the PMMA between the insulated
electrodes can also be dissolved
and leave the electrodes able to
of one
move independently
another.
2.2.3. Post-TDP processing
It takes about 50 minutes to remove the PMMA cladding which is about -500 pm in length, but
the fiber could remain in the solvent for more than a day to ensure a complete removal of only
this layer and not worry about the electrodes and their insulating polymer. Once the PMMA is
removed, the electrodes are indeed independent from one another and instead of forming one
cohesive cone they are splayed in various directions.
2.3
Selective etching of metals
Solvents can also be used to remove metal. Strong acids, such as hydrochloric acid (HCl), can be
diluted in water for a more controlled etching and to reduce the damage caused to the polymer
22
(Fig. 2.3). First, the fiber should be polished manually to create a smooth surface. Then, it is
dipped in a solution of 11% HCl for 5 minute intervals. Between each interval, the fiber tip
should be rinsed in water and then checked under a light microscope to see if metal is being
removed. This can be repeated up to -4 times to create channels that are tens of Pm deep, while
still preserving most of the polymer. More than 4 cycles results in large cracks in the polymer
surface, and a significant deviation from the initial cross section shape.
A
B
C
Fig.
2.3:
Selectively
removing metal. (A) The
initial fiber, (B) placed
in a well of HCl and
the
on
depending
amount of time the fiber
is left in solution, (C)
metal is removed.
2.4
Characterization
2.4.1. Imaging
To carefully image the cross-section of each fiber, the devices were imaged using a JEOL SEM
6060 scanning electron microscope. The SEM was also used to analyze the fiber after etching
metal, before implantation and at any other point in the fabrication process.
2.4.2. Connectorization
To use the fibers to fabricate devices that can be used for electrophysiology, the electrodes of
each fiber must be connectorized independently. To do this, sections of fiber are cut and placed
on a glass slide. Most of the fiber is covered by a second slide, which is securely taped to the first
23
by using heat-resistive tape. A few cm of the fiber are left unprotected, and the slides are placed
in a plasma etcher (AutoGlow Plasma, power at 100W) for 1 hour, in 10 minute intervals or until
the respective cladding has been etched away. Because the PMMA-COC-SnIn electrodes are
already separated from one another, they do not need to be put in the plasma etcher.
A surface mount connector is soldered to a to a custom designed printed circuit board
(PCB). The exposed electrodes from the etched fiber are placed on the PCB board and are
manually separated into independent pads on the PCB. A small drop of conductive silver paint is
added to ensure contact between the particular electrode-pad pair. After all the electrodes have
been connected and the silver paint is completely dried, epoxy is added to fix the connections.
The final step for design 1 is dipping the rest (non-etched) of the fiber in THF for 25 minutes and
cutting the fiber to the desired length. For design 2, the tip of the fiber can be placed in acetone
for 40 minutes so that the electrodes are again independent. A thin layer of poly-(ethylene
glycol), PEG, can then be coated on each electrode so that they form a bundle. This is only for
pre-implantation: since the electrodes are thin, -15 pm in diameter, they are very fragile and if
left independent would break. Since PEG is bio-inert and soluble in aqueous solutions, it will be
removed after implantation.
2.4.3. Impedance Measurement
There is a range of frequencies associated with electrophysiological signals, so impedance is
measured over a sinusoidal frequency sweep (10 mV, 20-106 Hz) using a precision LCR meter
(HP4284A, Agilent Technologies). After the fiber was connectorized to a PCB, the connector of
the device was fit into a matching female connector. This second PCB had insulated wires
connected to each pad, such that each device pad had a corresponding wire which could be
connecting to an alligator clip of the LCR. The tip of the fiber was submerged in 0.9% NaCl
24
solution, as was a ground wire which was connected to another alligator clip of the LCR. 5 trials
of each electrode were run, and the results averaged to give information about standard deviation
and consistency of the impedance values.
It was important to ensure that no part of the PCB was submerged in the NaCl solution,
or else the set-up would be shorted. A LabView program was written to collect the Z'
(resistance) and Z" (capacitance) of each electrode, and the data was then exported to CSV file.
2.4.4. Soak Tests
To mimic the effects of physiological conditions on the performance of the electrodes, the
devices were left to soak in 0.9% NaCl solution for 2 weeks. A layer of paraffin was placed
across the top of the vial to prevent the salt solution from evaporating over time. Impedance
spectroscopy was collected at the following time points: 0 days (pre-soaking), 1 day, 3 days, 7
days, 10 days and 14 days. The data was collected at the same time, in the evening, to keep any
potential outside influences as consistent as possible. Additionally, before running each set of
trials, a 500 kQ resistor was run in the LCR to ensure the data was reliable.
2.5
Electroplating
Long-term biocompatibility of Sn and SnIn is unknown, but because of TDP restraints, metals
that are more commonly used in electrophysiology such as Au are unable to be drawn with
polymers. However, once the fiber is drawn and connectorized, its metal can be selectively
etched and a different metal with better SNR and biocompatibility can be plated into the well
(Fig. 2.4). By matching the mounted PCB connector to the female part, as is done during
impedance measurement, each electrode can be electroplated independently.
25
The function generator (Agilent, 33500 B Series) is set to a pulse waveform which has a
width of 50 ps, a frequency of 100 kHz, amplitude of 1.4 V, and an offset of 0.7 V. These values
can be adjusted to change the thickness and rate of deposition. The metal-etched fiber and a
copper wire are placed in cyanide-free gold plating solution (Neuralynx). To observe the effects
and trends of electroplated gold, trials were first run on large 800 pm diameter PEI-Sn fibers.
Plating at the above conditions for 28 minutes completely coats the fiber polymer-metal tip with
a thick gold cap. Reducing the time to 7 minutes results in coverage of just the Sn metal, which
is 300 pxm in diameter. The fibers with 10 pm electrodes need only 180 seconds to fill the wells.
A
Fig.
B
2.4:
electroplating.
are plated
impedance of
and improve
Schematic
of
(A) Gold posts
the
to lower
the electrode tips
biocompatibility.
Additionally, the gold plated
posts can hopefully (B) more
with
interface
intimately
neurons upon implantation.
An optical microscope can be used in between rounds of plating to check the progression
and coverage of the gold cap. Every time the fiber is removed from the plating solution, it should
be placed in water for 3 minutes to remove any salt residues and excess gold solution. This
improves the smoothness of the surface and ensures only gold is on the surface. Then, a
Kimwipe can be placed gently at the corner of the fiber to wick away all the water. Plating can
be repeated for as long as needed, and done for each electrode.
26
The SEM can be used as well to look at the microstructure of the gold plated tips. Energy
Dispersive X-rays (EDS) can also be used to analyze the surface composition to see how much
Sn remains at the surface and to ensure that the material being plated is actually gold.
To observe the metallurgical junction between the drawn electrode and the electroplated
gold, a confocal microscope can be put into reflectance instead of fluorescence mode. After 600
nm, gold is 40% reflective whereas tin is much more reflective between 500-630 nm. A red beam
then should resolve the topography of the gold, and blue and green lights can show this
difference in reflectance. By imaging the sample from the side and overlaying the signals from
the different colors, it is possible to see the interface between the two metals. In addition to
evaluating the junction, it will be easier to see the thickness of the gold cap. By imaging the
fibers after plating for various times, the time at which the gold plates as a post versus where it
starts to spread outwards can be identified.
27
3. Results
Following the procedures described in Section 2 of this work, I have fabricated two types of
metal microelectrode arrays using a two-step thermal drawing process. These fibers have been
further modified using selective etching and electroplating techniques. In this section, I describe
the full characterization of these devices. Furthermore, I also show applications of one of the
devices (Design 1) for the recording of neural in vivo.
28
3.1
Design 1
(The results shown in this section and Section 3.2 have been published in Nature
Biotechnology as part of the article, "Multimodal fibers for simultaneous optical,
electrical and chemical interrogation of neural circuits in vivo", of which I am a coauthor)
3.1.1. Cross-section
In Design 1 there are two polymers, PPSU and PEI, and one metal, Sn, which comprise the fiber.
The Sn is insulated by a thin layer of PEI, and PPSU is used as the sacrificial cladding because it
is soluble in THF while PEI is not. Fabricated with a two-step TDP, the final cross section of this
fiber is shown in Fig. 3.1A
Fig. 3.1: Selective etching of the sacrificial cladding. (A) The cross-section of the drawn fiber,
unetched. The lighter gray polymer is PPSU, and it is contains no electrodes so it would have no
role in recording neural activity. (B) The transition between the unetched and the etched fiber. It
is easy to see each individually isolated fiber in the etched portion. (C) The cross-section of the
etched fiber. There are 7 electrodes which are approximately the same size, which are each
surrounded by PEI. Since the PEI covers the entire length of each electrode, it means only each
tip would be active once implanted in neural tissue. Since each electrode is insulated from one
another, the channels are not shorted. Instead, they can each independently record.
Before TDP, the first preform was 1.5" in diameter and contained one electrode which was 6
mm in diameter, and after both TDP steps, the fiber was -450 pm in total diameter (Fig. 3.A).
There are 7 electrodes contained at the core, with diameters that were on average, 7 pm +/- 1 pm.
They were evenly spaced, 22 pm +/- 2 pm, from one another to form a dense array. After
29
selectively etching the PPSU, the microelectrode bundle diameter was 85 im (Fig.3.1C). This is
comparable to the thickness of a single human hair (Fig. 3.2).
Fig. 3.2: Etched PPSU-PEI-Sn fiber. The fiber, which contains 7 Sn electrodes, is
approximately as thick as a human hair.
3.1.2. Flexibility
Selective etching reduces the diameter fiber probe. The flexibility of the fiber, measured in a
single cantilever setup, is greatly increased during this size reduction as well (Fig. 3.2). A steel
microwire of comparable diameter (-125 pm) was used as a control for flexibility since the
microwire is considered to cause little tissue damage.
80
Original fiber
-3
A
125 pm steel wire
Heartbeat rate
404)
2t-
rate
fRespiratory
Etched fiber
100
111
101
Frequency (Hz)
Fig. 3.3: Flexibility of the probe. The unetched (blue), and etched (red) fibers
are compared to a steel wire (black) of comparable diameter.
-
-
"Paim -
-
-
30
3.1.3. Impedance
Since only the tip of the electrode will be used in neural probing, it is important to measure its
impedance. Impedance spectroscopy in the range of 20-106 Hz was performed on multiple
electrodes in different sections of the fiber to obtain an average and standard deviation of
impedance. This served to evaluate the consistency of the devices and connectorization
procedures. We found Sn electrode impedance in a range from 0.7-1.3 MO at lower frequencies,
which decreased at higher frequencies (Fig. 3.4).
1.41.3~1.o~0.80.7
-0.6-
0.10
1
1
13o1
1 ol I tIII11
03
Frequency (Hz)
10?
104
Fig. 3.4: Impedance spectroscopy of Design 1. An average over the 7 electrodes
is plotted, along with the corresponding standard deviation.
3.2
Chronic Implantations
(special thanks to Ulrich Froriep, Andres Canales, Ryan Koppes)
Following characterization and connectorization to PCBs, the 7 electrode devices were implanted
chronically into the mouse medial prefrontal cortex (mPFC) by two other lab members, Ulrich
Froriep and Andres Canales. The devices were then evaluated for their in vivo recording
capabilities and a summary of these results is shown below (Fig. 3.5).
31
Neural activity was recorded from freely moving adult male wild type mice (Fig. 3.5A).
After analysis of the electrophysiological data, spike sorting revealed two separable action
potential shapes (units) recorded with the same electrode channel (Fig. 3.5B). Similar recordings
were then performed in several animals and the average SNR of the devices was found to be
13 6; the maximum observed SNR was 20 (Fig. 3.5A. This is finding illustrates the promise of
our technology, as the typical recordings in mice currently exhibit SNR of 2-3.
A
750
500
0
-250
460ensals
C0
50
200
100
B
250
)0
PC3
50.
0
1
2
3
Time (ms)
4
Fig. 3.5: Neural recording data. (A) Spontaneous neural activity for single neurons,
recorded in a WT mouse. (B) By using PCA and k-means clustering, each trace can be
attributed to a particular unit and these show that (C) two units can be recorded from,
simultaneously.
32
'U
The biological response and biocompatibility of the fiber probes was evaluated against a
steel microwire of comparable size (this work was done by postdoctoral scholar Dr. Ryan
Koppes). Immunohistochemistry was performed on brain slices (obtained by me and Dr. Ulrich
Froriep) after 3 days and after 3 months of implantation, and the slices were stained for 4
antibodies: IgG was used to evaluate the breaching of the BBB while GFAP, Ibal and Edl were
used to highlight the activity of astrocytes, microglia and macrophages in the vicinity of each
device. The latter three collectively illustrate the glial scarring around the implant. In addition to
the lower intensity of each marker in the vicinity of the fiber implant, the diameters of the holes
left by our devices in the tissue were smaller than those left by standard steel microwires of
comparable size. Since fiber probes are highly flexible, we hypothesized that they are able to
move along with brain during micromotion, and thus cause less damage to the surrounding
tissues.
3.3
Design 2
While THF removes the PPSU cladding, the inner core has the 7 electrodes embedded in a PEI
matrix. Because the electrodes are all concentrated at the core, they would cover a small range of
neurons once implanted. If the electrodes were scattered throughout the PPSU, they would not
form a single bundle and would be able to spread out once implanted. Since THF is such an
aggressive solvent, it could affect the thin insulating layer of PEI during the process of selective
etching. Keeping in mind the TDP restrictions and the concept of orthogonal solubilities, a new
materials platform can be determined which requires milder solvents. This is why I chose poly(methylmethacrylate), (PMMA, strengthened UV-Resistant slab, McMaster Carr, Robbinsville,
33
NJ) is used for a variety of applications, and is known by lithographers to easily dissolve in
acetone (Fig. 3.6).
This fiber was also fabricated with a two-step drawing process. Before the second step of
the TDP, the PMMA was slightly less than 1.5" long and each insulated electrode was 1 mm in
diameter. After the TDP, the dimensions of the rectangular cross-section were -520jn x -700
gm and each of the 5 electrodes was -17 pm in diameter (Fig. 3.6, Fig. 3.7). Once the sacrificial
cladding was etched away, the electrodes were independent from one another and when placed in
a vial with a magnetic stir bar and water, they moved along with the flow of the liquid.
Fig. 3.6: Schematic cross-section of PMMA-COC-SnIn fiber. Once the PMIMA is
removed, the electrodes are all separated.
Fig. 3.7: SEM cross-section of the PMMA-COC-SnIn fiber. This PMMA matrix has 5 COC-
SnIn electrodes, and 3 of these are visible (marked by the yellow circles). On the right, a
magnified view shows a light gray ring of COC and inside this ring is a SnIn electrode.
34
3.4
Electroplating
3.4.1. Selective Etching of Metal
The concentration of the HCL and the duration of time that the fiber is in the acid both affect how
much metal is removed. HCl diluted to -10% appeared to effectively remove metal, while
avoiding significant damage to the polymer matrix (Fig. 3.8).
Fig. 3.8: Selective etching of Sn. (A) SEM of a 9 electrode, hollow channel fiber. The metal tips
have been etched back by HCl for 5 minutes, and (B) a zoomed-in version shows that the circular
exposed channel is evident. Both images show regions of polymer damage, so the acid is diluted
to minimize this effect.
3.4.2. Characterization
It is important to remember that since the area of these electrode tips is -40x larger than that of
the previously described designs, the impedance will also be much lower. However, the trends of
impedance between the plated and non-plated fibers still hold and for completeness, the tips of
both Design 1 and Design 2 were plated and the impedance compared for each materials system.
The morphology of the plated electrode tips was evaluated using the SEM. Tips were
imaged after plating for 3 minutes (Fig. 3.9A), and after intentionally overplating for 27 minutes
(Fig. 3.9B) in order to cover the entire surface with Au.
35
Fig. 3.9: Gold-capped electrodes (A) after electroplating for 3 minutes. This image is zoomed in to
focus mostly on where the Sn tip is. The textured center of the picture, outlined mostly in red,
represents where the Sn is and also where the plated gold is mostly concentrated. (B) After
electroplating for 27 minutes, using the same voltage and pulse-width. In this image, however, the
entire view of the fiber is shown.
EDS was also done to analyze the composition of the surface to see what material was deposited.
Fig. 3.10 illustrates that, indeed, the electrode tips were coated with gold following the
electrodeposition procedure.
0~~~zoo
4M,3
31
*OD20
SM.
00
.0TOO A.OQ6
0
Fig. 3.10: EDS compositional analysis. This shows that the material plated on the surface is
indeed gold, as shown by the intensity of gold at the electrode tip and also by the number of
counts. Because the anode was copper, some ions were probably released in solution. This
particular fiber was not rinsed in water after plating so some of the copper could have dried on
the surface of the electrode.
36
The electrochemically deposited gold was found to form a very porous surface on top of the
electrodes (Fig 3.11). The degree of porosity could be reduced slightly by wicking away the gold
plating solution with a Kimwipe immediately following the deposition, but this procedure was
only effective at preventing the formation of larger pores. It is not clear, however, that the
porosity is an undesirable quality for a neural recording electrode, as a larger surface area of a
the
porous electrode yields lower impedance and potentially provides improved contact with
tissue.
Fig. 3.11: Porous gold surface. (A) A detailed view of the surface with a SEM shows many pores
of different sizes, with even some cracks. (B) At one portion of the fiber, there was no plated
gold and the thickness of the gold cap can be determined. (C) The surface, observed with a light
microscope, is rough and has a lot of texture. It is clear that the surface area of the tip is greatly
increased and this should increase the SNR of recordings.
Using a confocal microscope, the metallurgical junction between gold and SnIn was examined.
Fig. 3.12: Metallurgical junction. The side view
of the PMMA-COC-SnIn fiber is shown and the
edges of the fiber are outlined by the dark black
lines. The middle light gray section shows
where the electrodes are contained, and in this
section of the fiber there is just 1. In this image,
the images from the red and green beams are
overlaid. The SnIn is illuminated by green, but
at the tip, there is a yellow color which
represents the plated gold. Interestingly, it
seems that the gold plated on the sides of the
SnIn.
37
When gold is electrodeposited on the SnIn surface, the process is a bit different than that of Sn.
The electrode is mostly In, so after plating it for 3 minutes the surface is not completely coated
with gold (Fig. 3.13A). In Fig. 3.13B, some salt remains from the plating solution but the
microstructure of deposited gold is evident under this layer.
Fig. 3.13: Electroplated gold on SnIn. (A) This fiber is after the first drawing step of the PMMACOC-SnIn fiber. There is one SnIn electrode, and after electroplating with the same gold solution
for 3 minutes, half of the electrode is virtually uncovered and the other half has a mesh of
deposited gold. (B) Zooming-in on this plated half shows the leaf-like granules of the gold
crystals. The white layer on top is most likely from residual gold plating solution that dried and
left salt.
3.4.3. Effects on Impedance
The effects of gold plating on impedance were tested in three materials system: PEI-Sn single
electrode, PMMA-COC-SnIn single electrode, and 3 electrodes in a PPSU-PEI-Sn fiber which
are shown in Fig. 3.14, 3.15 and 3.16, respectively. In all cases, the impedance of the electrodes
was lower for the gold tip. Furthermore, the standard deviation of the impedance for the goldcoated electrodes was significantly lower than that for nonplated electrodes. This indicates that
this procedure yields a more reliable and chemically-stable electrode surface.
38
.10
ptplating
-preplating
6
9.4
E
E"
2
Fequency (Hz)
Fig. 3.14: Impedance spectroscopy for a 7 electrode PPSU-PEI-Sn fiber. Plating the
electrode tips reduces the impedance significantly and also decreases standard
deviation.
2500
-non-plated
5M00
10?
Frequency (Hz
10
10
Fig. 3.15: Impedance spectroscopy for a -500 pm electrode in a PEI-Sn fiber. The
curve shown is an average over 5 trials. As in Fig. 3.13, the standard deviation for the
plated fiber is much less than that of the non-plated fiber. One difference between the
two figures, however, is the magnitude of the impedance which as mentioned before is
due to the 100x increase in electrode diameter.
39
-::Plated
non-plated
40003500-
~3000
00
~2000
1000
500
Freppmney'00
Fig. 3.16: Impedance spectroscopy for a -300 pm electrode in a PMMA-COC-SnIn
fiber. The SnIn electrode is slightly smaller, which explains a slight increase in
impedance values. The curves are averaged over 5 trials for each sample, and both the
impedance values and standard deviation is substantially lower for the plated samples.
3.4.4. Soak tests
To mimic the physiological conditions the electrodes would be subject to, the fibers were soaked
in 0.9% NaCl solution for up to 2 months. Tip impedance of these soaked electrodes was
measured over 14 days to evaluate the stability of these values. The impedance at the following
time points was taken: 0 (pre-soak), 1, 3, 7, 10 days. Five trials of each fiber and each electrode
were run, and the data was averaged for each fiber and each electrode to watch the progression of
the impedance over time; the standard deviation was also plotted to see the reliability of the
impedance measurements.
After 2 months, the fiber tip was imaged with the SEM to see how the gold plated tip was
affected. Fig. 3.17 shows an elevated amount of salt at the location of the single gold-plated SnIn
electrode. This implies the gold remained and was the most densely charged portion of the fiber.
40
Fig. 3.17: SEM after soak test. There is an
elevation of salt at the center of the fiber, which
is where the gold-plated SnIn electrode is. The
surface area has been greatly increased, and
most of the fiber is covered by salt.
The quantitative effects from the soak tests are shown in Fig. 3.18.
Devte 1
14000
p do
dO
dl
-np
-p
12000
np d!
-
p d3
10000
-np
c13
P d7
np d7
p d1O
8000-
I
np dlO
6000
4000
200014k
10 1
10
10
10
Frequency [Hzl
10
10
10
Fig. 3.18: Soak tests for a -300 pm electrode in a COC-SnIn fiber. The curves in pink-red
hues represent plated ('p') fiber trials, while the blue scale represents non-plated ('np').
Each color represents a different day ('d'), and there are 5 time points shown here: 0 (presoak), 1, 3, 7 and 10. The plated fibers are -stable for all of the days, while the impedance
of the non-plated samples has a noticeable increase over time.
41
Another way to visualize the data from the soak tests is to compare the impedance of one plated
and one non-plated fiber, from the same time point, over a frequency sweep. Additionally, the
impedance for a particular device can be plotted over all the time points (Fig. 3.19).
B
A
80W[
Day 0
od
91
-
E 4000
2.0
g 4000-2000-
-'4-
I
T
--.
-
500
10
1
Frequency
Hz
2 4
8
8
Tme (days)
10
12
Fig. 3.19: Soak tests, replotted. (A) Impedance, as a function of frequency, for a plated (red) and
non-plated (blue) fiber. The time point was at 0 days, or before soaking. It is worth noting that
the shapes of the curves are much different: the plated looks like decay, but the non-plated curve
has two separate decay curves separated by plateau regions. (B) Impedance as a function of time,
with all the points at 1000 Hz. The plated gold impedance is lower and more stable.
42
14
4. Discussion
In this section, I summarize the results of fabrication and characterization of fiber-based
electrode arrays used for neural recordings. Furthermore, I assess the limitations of the fiberbased neural probe technology. Finally, I outline the future applications as well as new postprocessing techniques that could be implemented in conjunction with the fiber-based fabrication
of neural recording devices.
43
Conclusions
4.1
In this work, I demonstrated how after a fiber is drawn using TDP, its size can be further reduced
via selective etching. The materials can be chosen such that the outer-most cladding of the fiber
exhibits different solubility properties from those of the other components and thus can be
selectively etched away. Fibers can be fully electrically characterized and connectorized in
preparation for implantation. The devices are able to record neural activity from freely moving
mice with record-high SNR. Additionally, using selective etching metal can be removed to create
micro-wells. Then, electroplating can be used to deposit gold at the tips of each recording
electrode. This increases the stability of the recordings, as well as significantly decreases the
impedance of the electrodes. The plated gold exhibits high porosity, which provides a favorable
surface for interactions with neurons. An increased surface area may yield a more intimate
connection between an electrode and a cell, which is hypothesized to increase the SNR of
electrophysiological recordings.
Limitations and Future Work
4.2
For truly high density neural recordings and for a dynamic mapping of the brain, the number of
electrodes that can record neural activity must be significantly increased from 7 shown in this
thesis (or even 39 electrodes, as demonstrated by Andres Canales in his MS thesis). This can be
accomplished by incorporating an additional TDP step, applied to a preform comprised of
bundles of multi-electrode array fibers.
However, a significant challenge of our current fabrication process is the method for
connectorization of the electrodes to the PCBs. The current technique of moving 10 pn
electrodes by hand and using silver paint to secure the connection to a conductive pad is very
44
inefficient. Because these electrodes are so thin and fragile, it is very easy to break them during
routine handling and manipulation. Thus, this manual method of establishing electrical
connectorization often results in a poor yield of individually connected electrodes.
By designing chips with patterned electrodes to match the cross-section of the fiber, the
two can be aligned and then direct bonding techniques can establish secure connections.
Alternatively, a chip with an excess of small pads can be employed and electroplating techniques
can be used to connect the electrodes to the pads probabilistically.
As we have shown, the devices with Sn electrodes have successfully recorded neural
activity and exhibited good biocompatibility. The fibers based on SnIn electrodes must also
undergo a similar evaluation process. We aim to compare the performance of these new
generation fibers to our existing devices, as well as contrast recording ability of the electroplated
electrodes with those left untouched. These experiments will be performed in the mPFC of freely
moving mice across multiple time points. The SNR and the number of unique recorded action
potential shapes will quantify the chronic device performance in these studies. Additionally,
histological evaluation of the devices with and without gold plated tips will be used to correlate
the tissue response to the recording performance and contrast the interface properties of these
materials systems.
In this thesis, only electroplated gold tips have been investigated and characterized.
However, IrOx is a material that is becoming increasingly popular in neural interface
applications (Almquist et al, 2011). IrOx can be deposited at the tips of electrodes either using
electroplating or electroless deposition techniques (Sviridov et al, 2003). To further control the
surface, gold posts can be deposited and then capped by a layer of IrOx. This is advantageous
because of the differences in deposition kinetics between gold and IrOx- gold plates layer-by-
45
layer, whereas IrOx fills the edges before filling the center. Hence electrodeposition of gold can
be used to first increase the effective area of the device, which can then be followed by a few
layers of IrOx, which may ultimately yield a higher SNR due to improved interface with
neurons. Finally, additives can be suspended into the material that will be plated, which would
open the possibility for further optimization of multifunctional interfaces.
The multielectrode metal neural probes currently can only record neural activity, but they
cannot stimulate cells simultaenously. Thus, additional functionalities such as drug delivery and
optical stimulation capabilities integrated within a single device would reduce the number of
surgeries and the number of implants per animal. At the same time, it would increase the
complexity of experiments that can be done with the devices, and would potentially provide
answers to systems neuroscience questions. Optogenetics (Appendix) is a tool developed by
neuroscientists in the last decade to precisely control the activity of neurons that are made
optically sensitive by the means of genetic modification with light-gated membrane proteins,
microbial rhodopsins (Boyden et al, 2005). In the optogenetic experiments, optical pulses can be
delivered through a fiber core and the optically-evoked electrophysiological response would
mark the class of manipulated neurons. This information can be used to illustrate connectivity
between different brain regions and even individual neurons within the brain. Using the lessons
learned within the scope of this thesis project, we aim to extend the capabilities of our multielectrode arrays to optical transmission. Because PMMA is very transparent in the visible range
and has a high index of refraction, it is often used in polymer waveguides that are defined
lithographically. In our experiments, a high refractive index contrast between PMMA and COC
would enable the use of this materials pair for waveguides that are integrated within the fiber
probe.
46
Similarly to electrodes and optical waveguides, hollow channels can be incorporated
within the preform. These microfluidic features can then be used to deliver drugs to inhibit or
activate local regions of the brain. This information can be used as another way to reconstruct the
circuitry of the brain that does not require genetic modification. The response of cell types to a
particular drug could be used to analyze therapeutic potential of various neuromodulatory
compounds.
47
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6. Appendix
A.1 Optogenetics
Implanted electrodes have sufficient precision for recordings. To allow for equal specificity in
the stimulation front, neuroscientists in the last decade have developed optogenetics.
Optogenetics incorporates light-activated ion channels from the algal family into the membranes
of neurons.
B
55~2
3 .5425
Fig. A.1: (a) Ion channel of channelkhodoposin (ChR2). Blue light is absorbed by the channel to
push through a concentration of Na, Ca and K ions. This allows for on-demand excitation and
inhabitation of neurons that are transfected with ChR2 in their membranes. (b) The spectrum of
ChR2 is shown here, and it is clear that the channel is activated by blue light (-453 nm).
This tool allows for on-demand excitation and inhibition of neural activity from a particular class
of neurons. Each -odopsin is an ion channel which involves the transfer of different kinds of
ions. Because each ion type requires a different amount of energy to pass through the channel,
different -odopsins are sensitive to different particular wavelengths of light: for example,
halorhodopsin is activated by yellow light, while channelrhodopsin is activated by blue light. By
incorporating these channels into neurons, devices with light components can communicate with
51
the surrounding transfecte neurons. Since the development of optogenetics, engineers have
redesigned their devices shown in Fig. 1.1 and Fig. 1.2, and have even created new probes.
A.2 Selective etching for a waveguide system
The selective etching techniques described for the PPSU-PEI-Sn and PMMA-COC-SnIn
composites can also be applied to other materials systems which have additional functionalities.
For example, this procedure can be applied to fibers composed of polycarbonate (PC) claddings
with a COC core. The outer sacrificial cladding can be removed by dichloromethane. This
process occurs in 20 minutes, but there is a layering structure with PC also in the very center.
This means it is especially important to epoxy the ends of the fiber in order to preserve the
waveguide. Again, this selective etch allows for further reduction of the fiber diameter while
providing for a stable TDP.
Fig. A.2: Selective etching of the waveguide system. The PC is removed by dichloromethane.
Every preform can be post-processed to etch away sacrificial cladding layers, reducing the
overall size of the fiber while maintaining stable, micron-scale features in the TDP.
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