University of Canterbury
Department of Mechanical Engineering
Doctor of Philosophy
Automated Cellular Delivery and Surgery Device using
Date of Submission:
July 29, 2010
SUBMITTED BY: ASB
Mechatronics Research Lab, Room C230
SUPERVISOR: Wenhui Wang
Mechanical Engineering, Room E511
1. Introduction............................................................................................................................... 5
2. Background and Literature Review........................................................................................... 5
2.1. Cellular Manipulation................................................................................................... 5
2.2. Cellular and Neural Delivery........................................................................................ 6
2.3. Silicon and Polymer Microneedles............................................................................... 9
2.4. Nanoneedles.................................................................................................................. 13
3. Micro-/Nano-positioning stage.................................................................................................. 16
4. Objectives, Hypothesis and Questions to be answered.............................................................. 20
5. Research Framework and Methodology.................................................................................... 22
6. Gantt Chart................................................................................................................................. 32
7. Miscellaneous ............................................................................................................................ 32
2.1. Publication Plan............................................................................................................. 32
2.2. Research Supervisory Team.......................................................................................... 33
2.3. Equipments to be accessed............................................................................................ 33
It took me almost 6 months to come up with a research proposal from an idea and the journey has
been exhilarating. And this would not have been possible, thanks to the support, understanding and
expertise of those around me. And especially i feel lucky to have landed to work with Wenhui
Wang, my primary advisor whose intellectual rigor and personal commitment to Cellular
Engineering, Control Systems and Image Processing have germinated the ideas that i have briefly
described in this proposal and smoothed my rough edges as an evolving researcher.
My special thanks to the members of my supervisory team including Geoffrey Chase, Richard
Blaikie, Drusilla Mason, Yu Sun (University of Toronto) and Xinyu Liu (Harvard University) for
their kind consideration of advising my graduate research and the intellectual discussions that we
had. I am deeply indebted to the rest of Mechatronics Research Lab: Mervin Chandrapal, Shazlina
Johari, Robert Schattschneider, Syariful Syafiq Shamsudin, Craig Bennett, Michael Lang, Cecile
Muller, Stefan Schalk and Frank Rauer for their help, encouragement, guidance and particularly
friendship. Mostafa Nayerloo and Harminder Singh, in particular have been an endless source of
knowledge and invaluable friendly mentors. They are brilliant, yet humble and accessible, and
always willing to answer my questions, no matter how silly or stupid. Ashley Garill from the
School of Biological Sciences took time out from his busy schedule and helped me with some of
the microinjection techniques while Maan Alkaisi from the Department of Electrical and Computer
Engineering and Allison Downard from the Department of Chemistry shared their time for some
stimulating discussions regarding my research.
My special thanks to Anne Manuel for her assistance to me with all my paperworks, Julian Murphy
without whose help my preliminary experiment would never have been set-up coupled with his
expertise with Labview, Bruce Sparks who taught me the first steps in Solidworks and because of
whom all the Solidworks model in this proposal were made possible and Adam Latham and Paul
Southward for their assistance to me in acquiring a computer system for my personal and research
work. I am also grateful to Ross Millichamp and Dirk Barr for their generous donation of the
salmon fish eggs for my preliminary experiments.
Outside my research, some of the persons who deserve special mention are David and Rosemary
Troughton and Peter Hallinan and Patricia Allan for their extraordinary help and those wonderful
piano lessons, Russel and Ivy James for organizing those amazing Operation Friendship meets that
brought me in close contact with people from across the globe, Sarah Beavan and Lawrence Teo at
the International Student Support Office for their friendly guidance with my research matters
ranging from ethics to proposal format and documentation and finally Farida Memon at the
International Office who was my first contact while i was applying for my graduate admission at
I would like to thank all my friends for their support over the years. But i would not be writing this
proposal if it weren't for two very important persons in my life, my father and mother. Had they not
encouraged and instilled human values in me, i would have never come here. I would also like to
thank my younger brother, Anirban for his uncanny love and i wish he works hard as much as he
can and achieves success that he deserves. Because of limited space, i may have left out the names
of some people who contributed along the way. If so, i am sorry, but please realize that i really do
appreciate all your efforts and support very much. Funding for this work has been generously
provided by the Department of Mechanical Engineering through the Premier PhD Scholarship.
Finally, all mistakes in this research proposal are mine.
Cells are densely packed with thousands of integrating components that must be produced,
transported, assembled into complexes and recycled all at the appropriate time and place. While
large-scale systematic characterization of the components of cells or other biological samples have
become possible, nonetheless modern technologies doesn’t allow observation of the spatial and
temporal organization of these entities while they are at work in cells. There is a dearth of
experimental tools that can analyze the cells’ complicated internal complexes. In this direction,
molecule screening and DNA sequencing technologies, is critical in molecular biology and drug
discovery. Molecular Screening requires that target molecules be introduced into cell arrays to
permit cellular-function-targeted molecules to directly regulate cell development and their functions
to be quantified. DNA sequencing involves the ability to sequence individual genomes, or relevant
portions of genomes.
There has been an increasing interest in the last decade towards cellular manipulation for drug
delivery, gene therapy, intracytoplasmic sperm injection (ICSI) among others for drug discovery
and research and for a deeper understanding of human development and disease. While manual
microinjection using conventional glass pipettes remain to be the most widely used technique for
cellular delivery, it has a very low throughput time coupled with increased human fatigue. Albeit
alternative contact and non-contact microinjection techniques have been developed including viral
vectors, electroporation, liposomal carriers, laser trapping among others, each of them has several
short-comings thus making them less attractive for cellular injection.
Thus the main idea of this research is to develop an automated cellular delivery and surgery device
using micro/nano fabrication technology that can inject foreign substances like proteins, nucleic
acids, DNA, Quantum Dots among others into the parallel arrays of cells with a high throughput
rate and flexibility. While single cell manipulation seems to be the major focus, it is gradually
taking a paradigm shift to parallel single-cell manipulation for very high throughput processes. The
automation would involve the development of a 2-axis microstage-needle assembly device,
exploring simple yet efficient methods of sorting and trapping the cells and development of control
and image processing algorithms to aid the automation process.
The anticipated duration of the project is approximately 3 years.
Cell manipulation in a precise and dose-controllable manner has been increasingly used in drug
delivery, toxicology, functional genomics and various other fields of biology, medicine and
biomedical engineering. This includes pro-nuclei deoxyribonucleic-acid (DNA) injection,
intracytoplasmic sperm injection (ICSI), gene therapy etc. for investigating specific cellular
responses. Moreover recent advances in microbiology including cloning signify the fact that
increasingly complex manipulation strategies for manipulation of biological cells are needed. The
importance of cell manipulation is highlighted by the following two examples.
• ICSI is an in vitro fertilization procedure for animal or human reproduction in which a single
sperm is injected directly into an egg. Since its first introduction by Palermo et al.1, it has been
widely used across the world. Refer to Figure 1.
Figure 1: Oocyte injected during ICSI
• In the case of gene therapy, normal genes are inserted into the human cells and biological
tissues to replace an abnormal, disease-causing gene thus treating dreadful disease like cancer
where harmful mutant alleles are replaced with functional ones. It was first conducted on a four-
year-old child suffering from a rare genetic disease called Severe Combined Immunodeficiency
on September 14, 1990, by a group of physicians including W. French Anderson and his
colleagues R. Michael Blaese, C. Bouzaid and Kenneth Culver from the U.S. National Institute
Nanoparticulate pharmaceutical agents can be injected into tumorous cells which can then be
stimulated externally using electromagnetic forces to emit heat and thus the tumor cells get
destroyed by the stimulated particles. Micro/nano-needles can be injected into living cells to deliver
drugs or genes and then their behaviors and characteristics can be studied or nanoparticulate
steroids are introduced into the body’s own red blood cells; as the cells die their natural deaths, the
steroids are released to the body in very small doses, thus minimizing, if not excluding the side-
effects of many steroid treatments4. These and many more examples, some of them already
developed and some in the process of development are great human ambitions to eradicate dreadful
diseases like Cancer, Parkinson's disease, Alzheimer's disease etc. On a broad scale, these
ambitions can be divided into four broad areas including theranostics (combination of diagnosis and
therapeutic functionality in one device, enabling pre-symptomatic treatment), polymer therapeutics
(rational design of nanomedicines), targeted drug-delivery (individualized medicine) and
regenerative medicine (cell repair).
2. Background and Literature Review
2.1. Cellular manipulation
Cellular manipulation involves such operations as positioning, grasping and injecting foreign
substances into the cells. For example, introducing deoxyribonucleic acid (DNA) or transgene into
the nucleus of a cell where it is randomly inserted into the host genome for creating knockout mice
are technically challenging. The transfection and survival rate are approximately 20%5. There are
various techniques for gene delivery including viral vectors, electroporation and liposomal carriers6.
For example, viral vectors involve the viral particles that encapsulate a modified genome carrying a
therapeutic gene cassette coupled with large DNA or RNA molecules in place of the viral genome
that they introduce into the cells7. Thus even if the stable transgenic organism is created, it would
have some of the viral genome integrated as well. It also limits the maximum size of the delivered
transgene, thus limiting the amount of flanking DNA and regulatory elements introduced into the
cell. The lack of these regions may reduce nuclear localization, chromosomal integration and
expression8. Electroporation is another physical technique of importing small molecules and
macromolecules into cells via increased cell membrane permeability9.
In general, there are several technologies for introducing foreign materials into a cell that can be
broadly classified into optical, electrical, magnetic and mechanical micromanipulation. All the
methods as discussed above share some common disadvantages. They are harmful to cells to some
extent and methods like laser trapping10 may induce abnormalities in the cells' genetic material.
Again methods like electric-field-induced rotation of cells11 is not feasible due to the lack of means
of cell holding in place for injection, as the magnitude of electric fields has to be kept low to avoid
damaging any cell.
While all these techniques have their own merits and demerits, microinjection with a single glass
micropipette remains the most effective in terms of cell damage, viability, waste and effectiveness
of delivering foreign materials, specificity and freedom from concerns about phenotype alteration.
Marshall Barber was the first who developed the microinjection technique using pipettes to isolate
bacterial cells12. Nonetheless, testing cellular responses to molecular targets and obtaining
statistically significant data demands injecting thousands of cells within a very short span of time.
Thus, manual injection is not only slow, but it also causes human fatigue due to the fact that it is
very laborious and thus directly affects performance and success rates. In addition, the latest
advances in micro/nano-fabrication technology [i.e. micromachining or Micro/Nano-
Electromechanical Systems (MEMS/NEMS)] that uses tools developed by the microelectronics
industry to develop integrated circuits (ICs) have had a major impact on drug and gene delivery.
2.2. Cellular and Neural Delivery
In spite of several non-contact biomanipulation techniques available, the contact manipulation
using microinjection remains one of the most prominent biomanipulation methods for loading cells
that can inject large number and variety of macromolecules to a wide variety of cell types coupled
with high cell viability. Microinjection is one form of cell manipulation as the direct pressure
injection of a solution into a cell through a glass capillary which is an effective and reproducible
method for introducing exogenous material into cells in culture13. The molecules of interest include
peptides, proteins, oligonucleotides, DNA and a variety of other substances that alter or assay cell
function. Nonetheless single-cell manipulation involves injecting one cell at a time with individual
glass micropipettes observed under an optical microscope, which makes the task not only laborious
and causes human fatigue, but also it is viable for only a small number of cells, for example less
than 100 cells. There have been various efforts in automating cell injection including visually-
servoed system, semi-automatic and tele-operated systems.
In automatic biomanipulation of cells or micro-assembling micro-components, actuators with high-
precision such as piezoelectric driven manipulators are used14. Nevertheless, most of the current
microactuators, based on electrostatic, thermal, magnetic or pneumatic principles are not suitable
for operating in liquid surroundings. Sun and Nelson15 developed a microrobotic system capable of
performing automatic embryo pronuclei DNA injection autonomously through a hybrid visual
servoing control scheme that achieves a success rate of 100%.
Figure 2: Automatic cell injection system. microrobot-A and microrobot-B, which are three-degrees-of-freedom
motorized micromanipulators with a travel of 25 mm and a 0.04 µm positioning resolution along each axis, control the
position of embryos and micropipette, respectively. The system obtains visual feedback through the camera and
microscope. The computer-controlled pico-injector provides positive pressure for material deposition16.
Figure 3: Illustration of the automated injection flow. Top row: 3-D view. Bottom row: microscopic (image) 2-D view.
(A) The vertical height of the micropipette tip is determined with a computer vision approach. This step is required only
once at the beginning of one batch. (B) Micropipette at the home position. The white curve outlines the recognized
cytoplasm contour. The white dot represents the cytoplasm center. (C) Embryo is brought to the center of the field of
view. Micropipette is positioned at the switching point. (D) Micropipette tip penetrates the embryo and deposits
materials at a pre-set destination in a specified volume. (E) Micropipette retracts out of the embryo. (F) Micropipette
returns to the home position, and the next embryo is brought into the field of view. From (B) to (C), and from (E) to
(F), the two microrobots move in parallel to increase injection throughput16.
Advancing with the above previous work, Wang et al.16 developed a microrobotic system for fully
automated zebrafish embryo injection integrating computer vision and motion control as shown in
Figures 2 and 3. It achieves a high throughput rate of injecting 15 zebrafish embryos per minute
with a 98% survival rate, a 99% success rate and a 98.5% phenotypic rate. Multiple bioactive
foreign compounds such as plasmids, RNAs, antibodies, peptides, diffusion markers, elicitors etc.
can be introduced into the same target single-cells17. A computer controlled piezo-manipulator
system for biomedical applications such as intracytoplasmic sperm injection (ICSI) was developed
by Tan and Ng18. A micromachined system capable of controlled DNA injection into the cells was
described by Chun et al.19. The injection system is composed of two components – the hollow
micro-capillaries for injection and the micro-chambers for cell trapping. Anis et al.20 presented the
preliminary results of the development of a fully automated workstation for single cell
manipulation. It is capable of automated selection and transfer of individual living cells of interest
to analysis locations.
A single-cell manipulation supporting robot (SMSR), which enables a high throughput
microinjection was developed by Matsuoka et al21. It is a semi-automated machine which would
prove to be a supporting machine for both experts in microinjection and beginners. SMSR was
applied to the microinjection into rice protoplasts and mouse embryonic stem (ES) cells. Albeit this
was the first published report about successful microinjection into ES cells and the level of
throughput achieved was 17 times faster than that of the robot-less work, the success rate was very
low. In case of rice protoplasts, it was 5-10% (for non-adhesive cells) and 7-8% (for adhesive cells)
and in case of mouse ES cells, it was just 0.2-2.2%. A force feedback interface capable of
measuring µN – mN forces and simultaneously provides a haptic display of the cell injection forces
was developed by Pillarisetti et al22. PVDF piezoelectric polymer film was used to develop the
force sensor because of excellent sensitivity, high compliance and high signal-to-noise ratio. The
force sensor was integrated with the Biomanipulation system to detect forces in real time.
Experiments were performed on flying fish and salmon fish egg cells. The average force values
were found to be 1.6075 mN and 2.2694 mN respectively. This was followed by human factors
studies to evaluate the combined effect of vision and force feedback on cell injection outcomes,
rather than vision feedback only23. The findings confirmed that a system with combined vision and
force feedback capability can lead to potentially higher success rates in transgenesis, specifically
where mechanical manipulation techniques are involved.
Nevertheless there are several challenges in the automatic microinjection of single adherent cells24.
The volume injected can be influenced by parameters such as applied injection pressure, the
application time of the injection pressure and the level of the balance pressure. Either there is an
undesired efflux from or influx into the micro-capillary. Some of the parameters that affect the
hardware include size of the tip opening, tip length, surface treatment and possible tip breakage for
micro-capillary tip, stability of the pressure source, accuracy of the pressure regulator and the speed
of the check valve for the microinjector and positioning accuracy and preciseness of the axial
movement for the positioning and penetrating device.
In addition, neural recording from the animal brain to correlate neural activity with external
stimulation and behavior has been one of the primary focus of interest for neuroscientists for
decades. Neuronal communication is at the core of brain activity, and understanding its signaling is
the key to understanding the functions of the brain. For more detailed understanding of the neuronal
activities and functions, the readers may refer to the excellent review paper by Hanein et al.24 Over
time, there have been the developments of a wide variety of electrode-based-needles to study
neuronal signaling. Some are small, localized needle with a sharp and insulated exposed tip
consisting of a conducting wire; others include glass capillary tips with sub-micron dimensions
filled with an electrolyte (e.g. KCL) and consisting of Ag/AgCl electrode in the electrolyte etc.
Nonetheless, the fundamental disadvantage in all of these recording electrodes is that they consist
of a metal-electrolyte interface, which makes the system rather complex for multi-site recording25.
This is where MEMS neuronal needles are particularly promising due to their small dimensions and
the ease with which multi-site devices can be produced. Some of the most popular types of needles
for cellular, systemic and neural delivery have been discussed in the next section.
2.3. Silicon and Polymer Microneedles
Microneedles fabricated using MEMS technology that are made up of silicon, noble metals and
dielectric layers such as silicon dioxide, nitride or polyimide, can be divided into two major
categories; in-plane needles where the needle shaft is fabricated in a plane parallel to the substrate,
and out-of-plane needles which have their shafts perpendicular to the wafer plane. While some of
these in-plane needles allow for integration of electronics, nonetheless, since they can only be
arranged in single rows and problems associated with interconnects make them very challenging to
integrate with self-governing microsystems. On the other side, the out-of-plane needles can be
arranged in arrays so that fluid can be delivered or sampled over a wider area making the system
more stable and robust25.
Figure 4: Images of multi-needle glass and polymer microneedle arrays. (a, b) Glass microneedles assembled into an
array using epoxy. (c) Polyglycolide polymer microneedle and (d) poly-lactide-co-glycolide polymer microneedle array
molded from glass microneedle masters. Bars = 100 µm26.
The other major basis of classification of microneedles is the method of fluid/material delivery:-
solid and hollow microneedles. In terms of drug delivery studies, much attention has been paid
towards solid microneedles (Figure 4). They have been used either to pierce holes in the skin as a
pre-treatment before application of a transdermal patch or coated with drug that dissolves off the
needles upon insertion into the skin26. On the other side, hollow microneedles have received less
focus because they are more challenging to use. Due to their weak structures compared to solid
microneedles, they have additional constraints on needle design and insertion methods27. The
needle tip sharpness is reduced due to the placement of the bore opening at the needle tip and this
makes insertion into skin more difficult. Also the flow through the bore faces great resistance by
the dense dermal tissue beneath the microneedle tip and blockage owing to possible tissue coring
within the needle bore28.
Following the pioneering work by Wise et al.29, there have been numerous studies that exploited
integrated-circuit technology to build neurological microneedles. One such needle as shown in
Figure 5 was developed by Jingkuang Chen with Kendall Wise and others at the University of
Michigan, Ann Arbor30. This bulk-micro-machined multichannel silicon needle, incorporating one
to four flow channels are capable of selectively delivering chemicals at the cellular level and
electrically recording from and stimulating neurons in vivo. The needles bury micro-channels in the
needle substrate and are compatible with the formation of electrode arrays for electrical recording
and stimulation on the same chip, allowing multiple chemical delivery ports and multiple
recording/stimulating sites on a single shank.
Figure 5: (a) Neural probe designs having one, two, three, and four micro channels for drug delivery. (b) Overall SEM
view of a probe containing three drug delivery channels. Each outlet orifice has a recording site beside it. The site at the
tip of the probe has a large area and can serve both for recording and stimulation30.
Figure 6: (a) Concept of a miniature syringe (not to scale). Pressing a finger on the deformable drug reservoir drives
the needles into the skin and injects the drug suspension or solution into the epidermal skin layer. (b) A MEMS syringe
with 8 silicon microneedles and a PDMS drug container32.
Stoeber and Liepmann31 developed a disposable hollow out-of-plane silicon needle using a
combination of Deep Reactive Ion Etching (DRIE) and soft lithography bonded with a deformable
Poly Dimethyl-Siloxane (PDMS) reservoir for a suspension of lyophilized drug. The design avoids
clog formation in the needle channels and permits a density of 600 needles/cm² for shaft length of
200 µm. As an extension of their previous work in , Hafeli et al.32 presented a microfabrication
process for silicon microneedle based out-of-plane miniature syringes. The fluid-filled reservoir
consisting of the drug solution or microparticle suspension and made out of PDMS was bonded to
the silicon substrate consisting of the hollow needles as shown in Figure 6. Teo et al.33 fabricated
both solid and hollow microneedles with straight-side walls for transdermal drug delivery. The
solid microneedles were 130 µm high, 80 µm diameter and an interneedle distance of 200 µm. The
hollow microneedles were 150 µm high, an interneedle distance of 200 µm and an inner and outer
diameter of 25 µm/60 µm and 80 µm/100 µm respectively. Both types of needles were arranged in
a 10 by 10 array and the hollow needles were coupled to a conventional 1 ml syringe. The needles
were fabricated using a combination of photolithography and DRIE.
Trimmer et al.34 developed microneedles for injecting DNA into cells, having heights ranging from
10 µm to several hundred microns. They are pyramidal in shape with a half angle of about 13° and
the tip is ultra-sharp. It allows parallel cell injection in an orderly square pattern. The fabrication
was done using bulk micromachining including anisotropic wet etching. These microneedles were
used to inject DNA into tobacco leaves and nematode cells.
Figure 7: SEM of micromachined needle array. Individual needle channels are 2mm long and have center-to-center
spacing of 200 µm. The inner dimensions of each needle are approximately 30 µm wide and 20 µm high. The total
needle array width is 5.2 mm. Needle coupling channels are centered along the length of each needle are 100 µm
Brazzle et al.35 designed, fabricated and characterized a new type of fluid coupled hollow in-plane
metallic micro-machined needle arrays as shown in Figure 7. The fabrication process includes p+
etch-stop membrane technology, anisotropic etching of silicon in KOH, sacrificial thick photo resist
micro-molding technology and micro-electro-deposition technology. They fabricated an array of 25
needles with fluid coupling channels and dual structural supports. The needle arrays were packaged
using machined acrylic that serves as an interface between a standard syringe and the array36.
Continuing on their previous designs, they designed and fabricated a new type of hollow metallic
active microneedle. It includes design features such as tapered needle tips, multiple output ports on
the back and front of each needle, multiple lumens and multiple input ports and bioluminescence
based biosensors for monitoring metabolic levels.
Figure 8: Scanning electron micrographs (20 by 20 array) of microneedles made by the reactive ion etching
Figure 9: (a) Two silicon microneedles placed alongside a human hair. (b) Top view (optical microscope) showing the
tip end of a microneedle39.
Henry et al.37 developed one of the first solid out-of-plane micro-machined needles for transdermal
drug delivery which enhances transport of molecules across skin by more than four orders of
magnitude as shown in Figure 8. These microneedles were fabricated using a reactive ion etching
technique based on the Black Silicon Method developed at the University of Twente in
Netherlands38. The microneedles were fabricated as a 20 × 20 array with extremely sharp tips
(radius of curvature < 1 µm) and 150 µm long. They were also used on human cadaver epidermis to
test their transport-enhancing and mechanical properties. Lin and Pisano39 designed and fabricated
hollow in-plane silicon-processed microneedles which were integrated with bubble-powered micro-
pumps in the form of a series of polycrystalline silicon heater strips running across the floor of the
channels at the shank end as shown in Figure 9.
Figure 10: (a) SEM images of side-opened microneedles, the hole beginning at the base of the needle (b) SEM images
of side-opened microneedles, the hole beginning approximately 50 µm above the base of the needle. The length of the
structure is 210 µm40.
Griss and Stemme40 developed a hollow out-of-plane silicon microneedle array (21 needles) that
has openings in the shaft rather than having an orifice at the tip, as shown in Figure 10. The size
and position of the side openings are defined by process parameters and not by the specific mask
design. The fabrication is based on triple deep reactive ion etching (DRIE) and double
photolithography where vertical walls of the DRIE etched high aspect ratio silicon structures stay
vertical during an isotropic plasma etch. The microneedle array was assembled on a brass carrier
that was connected to fluid silicone tube. The needle was 210 µm long. Nordquist et al.41 showed
that microneedles are a possible treatment strategy for a common fast-acting insulin Lispro, which
requires minimal training and attention as shown in Figure 11. The microneedles were fabricated by
plasma etching of mono-crystalline silicon using MEMS batch processing techniques. The needles
were organized on 4 × 4 mm2 chips with 21 hollow needles. The needle array attached to a drug
dispenser, fabricated using similar micro-fabrication techniques, is designed to store and dispense a
drug volume at a certain flow rate. Its working principle is based on a thermally expandable silicone
material which expands into a liquid reservoir and thereby causing the liquid to move. The
expansion rate and thus the flow of the liquid, is controlled by the voltage supplied to the dispenser.
Figure 11: (a) SEM picture showing a close-up on several microneedles. (b) Assembled drug delivery patch. The unit
can store and dispense 12 ml of liquid41.
Park et al.42 demonstrated the fabrication, mechanical properties and transdermal drug delivery of
biodegradable polymer microneedles made out of polylactic acid (PLA), polyglycolic acid (PGA)
and their co-polymers (PLGA). Sharp tips were achieved by adapting micro-fabrication techniques
to produce beveled, chisel-tip and tapered-cone microneedles. The beveled-tip microneedles were
created by molding and replicating master structures fabricated by etching the tips of cylindrical
posts made of SU-8 photo resist. When inverted into human cadaver skin, polymer microneedles
were shown to create pathways for transdermal transport that increased skin permeability to calcein
and bovine serum albumin by up to 3 orders of magnitude. Ji et al.43 fabricated three types of
microneedle structures by isotropic etching in inductively coupled plasma (ICP) using SF6/O2
gases combination of isotropic etching with deep etching and wet etching. The microneedles had a
maximum height of 120 µm with square mask dimension of 80 µm, a center-to-center distance of
150 µm. The microneedle array with biodegradable micro-porous silicon tips was further developed
for drug delivery.
Reed el al.44 fabricated arrays of microneedles using anisotropic silicon etching for piercing
compressed plaque and delivering anti-restenosis therapies into coronary arteries as shown in
Figure 12. Zahn et al.45 developed a two-wafer polysilicon micro molding process for fabricating
hollow microneedles which are smaller, smoother and sharper with complicated geometries for
micro fluidic applications. The fabrication process included a single wafer process including
Reactive Ion Etching (RIE), LPCVD and DRIE.
Figure 12: Silicon microprobe fabricated by anisotropic silicon etching44.
Nanotechnology has infiltrated the field of cell biology in the form of quantum dots, nanofibers and
carbon nanotubes (CNTs), with applications ranging from imaging to tissue engineering. Among
these nanomaterials, Carbon Nanotube needles offer an attractive alternative to other microneedles
as discussed above due to their small size, high mechanical and structural strength and electrical
Chen et al.46 reported the development of a nanoscale cell injection system called the nano-injector
that uses carbon nanotubes to deliver cargo into cells as shown in Figure 13. A single multiwalled
carbon nanotube (MWNT) attached to an Atomic Force Microscope (AFM) tip served as the
nanoneedles and an AFM integrated with an inverted fluorescence microscope served as the nano-
manipulator. The nanoneedle diameter ranged from 10-20 nm. Qdot streptavidin was injected into
cultured HeLa cells. The Qdot streptavidin was attached to the MWNT surface and when inside the
cell, it released due to the reducing environment of the cytosol, thereby cleaving the disulfide and
liberating the cargo. Hara et al.47 developed a device called CellBee with an ultra-thin needle called
Nanoblade for pinpoint delivery of chemicals, proteins and nucleic acids into cultured cells. The
basic principle involves the flow of molecules from the culture medium into cells through a rupture
in the plasma membrane made by a needle puncture. DNA transfection into cultured HeLa cells is
achieved by stabbing the needle tip into the nucleus. The Nanoblade was fabricated by using
focused ion beam (FIB) technology by shaving the AFM cantilever AC160BN.
Figure 13: Characterization of nanoneedles before and after loading the cargo. (a) SEM image of a MWNT-AFM tip.
(b) TEM image of the tip region of A. (c) TEM image of a MWNT-AFM tip coated with linker 1 and conjugated with
Hoshino et al.48 proposed a membrane penetration process without any external force called “cell-
driven self-insertion” where the cell migration and adhesion forces can be employed for the
insertion of a nano-needle electrode into a live cell through its cell membrane without damaging the
cell as shown in Figure 14. The micro well structures on a glass plate had a low cell-binding
coating of 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer as an anti-biofouling
material, a coating of cellular adhesive molecules (fibronectin) and an electrically conductive
tungsten nanoneedle (190 nm diameter). The nanoneedles were self-consumable and disappeared
into the micro wells except their bases after 5 days.
Prinz and Prinz49 developed pilot models of micro-and nano needles and syringes based on
InGaAs/GaAs and Si/GeSi tubes suitable for intracellular microinjections as shown in Figure 15.
This work was an extension of their previous related work50 51. The approach is based on self-
rolling of a thin strained epitaxial heterofilm. Selective etching of a sacrificial layer provided
underneath the heterolayers frees the heterolayers from bonding with substrate and the resulting
film rolls in a tube. The tubes once inside the cell injected the chemical by a pressing and
deformation mechanism. The nanotubes had dimensions ranging from 5 µm to 50 nm, with ultra
thin walls (outer and inner diameters were almost same) and had good elastic and strength
Figure 14: Scanning ion microscopy image of a tungsten nanoneedle (190nm diameter and 8 µm height) on the bottom
of a 10-µm-deep micro well. The white arrow head indicates the boundary of the glass micro well and the magnetic
Figure 15: Tree coiled SiGe/Si needles with notch edges (diameter 7 m). The wall thickness is 35 nm49.
Chen et al.52 demonstrated a strategy for interfacing biocompatible CNTs with cell surfaces by
virtue of carbohydrate-receptor interactions. They coated CNTs with a bio-mimetic polymer
designed to mimic cell surface mucin glycol-proteins. The functionalized CNTs were then bound to
cell surfaces via specific carbohydrate receptors. Whereas, unmodified CNTs induced cell death,
the functionalized CNTs were found to be non-toxic. Pantarotto et al.53 studied the utilization of
carbon nanotubes as components for engineering a novel nanotube based gene delivery vector
system. They showed that ammonium-functionalized CNTs (f-CNTs) are able to associate with
plasmid DNA through electrostatic interactions. Upon interaction with mammalian cells, these f-
CNTs penetrate the cell membranes and are taken up into the cells. The observed nanotube has a
diameter of 20 nm and an apparent length of around 200 nm. F-SWNTs complexed with plasmid
DNA (charge ratio between the ammonium groups at the SWNT surface and the phosphate groups
of the DNA), facilitate higher DNA uptake and gene expression in vitro than could be achieved
with DNA alone.
Vakarelski et al.54 fabricated robust nanosurgical needles as shown in Figure 16, for single cell
manipulation by modifying multiwalled CNT terminated AFM tips and demonstrated their use to
carry nanoparticulate payloads and to penetrate the plasma membrane of living pleural mesothelial
cells at the indentation depths (100-200 nm) and penetration forces (100-200 pN). Kouklin et al.55
developed and employed a modified dielectrophoretic based technique for assembling individual
carbon nanotubes into highly aligned, nanoscale needles that are electrically and mechanically
interfaced to micro-needle bases as shown in Figure 17.
Figure 16: (a) SEM micrographs illustrating a typical AFM tip (MikroMash CSC38) with attached carbon nanotubes
bundle. Magnified images of the MCNT tip (b) prior to and (c) after sputtering with a 2-nm-thick carbon coating
followed by a 3-nm-thick layer of gold54.
They are electrically conductive and resilient to mechanical and biomolecule loading and can easily
penetrate cells both in vivo and in vitro due to their high aspect ratio. Obataya et al.56 developed a
system for living cell operation by using AFM and a modified AFM tip shaped as ultrathin needles
of 200-300 nm in diameter and 6-8 µm in length using focused ion beam etching. The system was
successfully on human embryonic kidney (HEK 293). Thus by modifying the surface of the needle,
various molecules can be loaded such as nucleic acids, proteins or chemicals through standard
Figure 17: SEM images of MWNT-bundle (left) coated with silicone as described in the text and SWNT-bundle
assembled from 1.5 µm long and 2 nm diameter arc-produced nanotubes (right). The inset shows a detailed view of a
typical MWNT bundle-electrode contact area with no coating applied, bar is 1 µm55.
3. Micro-/Nano-positioning stage
Indermühle et al.57 58 were one of the first research groups to have fabricated and operated a silicon
xy-microstage with integrated comb actuators for scanning displacement, a sharp tip for AFM
profiling and a via hole for optical detection. The fabrication was a combination of Reactive Ion
Etching and Silicon Fusion Bonding. The maximum displacement observed was 12 µm for an
applied voltage of 300 V and the microstage could be moved over an area of about 3 µm × 3 µm by
actuating two combs with voltages of 150 V. Tips as high as 55 µm with an aspect ratio of about
1.7:1 and tip radius of curvature as small as 10 nm were obtained.
Figure 18: Close up view of a large tip integrated on the central stage of an xy-micro stage58
Fan et al.59 reported a self-assembled, surface micromachined micro-XYZ stage with lateral
scanning of upto 120 µm and vertical scanning of upto 250 µm and fine positioning accuracy with
step resolutions of 27 nm, using scratch drive actuators (SDA). A group of nine SDAs have been
integrated with each actuator plate and the micro-stage is fabricated using the three-layer
polysilicon surface micromachining technology called Multi-User MEMS Processes (MUMPS). It
could be easily integrated with surface-micromechanical optical elements, such as micro-gratings,
micro-mirrors and refractive microlens. A two-depth, large displacement (160 µm × 160 µm),
highly linear motion, SCS XY micro-positioning stage fabricated by deep silicon etching and IR
front-to-back alignment was described by Lee et al.60 Four sets of comb-actuators drive the stage
each aligned at 90 degrees w.r.t. the adjacent actuator.
Figure 19: (a) The scanning electron micrograph (SEM) of assembled micro-XYZ stage59. Four micro-Fresnel lens
have been integrated with the XYZ stage. (b) Solid model of the two-axis microactuator61
The design, fabrication, modelling and control of a two-axis electrostatic micro-actuator for
precision manipulation tasks was described by Sun et al.61. The fabrication involves DRIE on
Silicon-on-Insulator (SOI) to form high aspect ratio suspending interdigitated comb drive actuators
which are suspended by completely removing the substrate beneath the comb-drive structure. The
authors developed a PID controller for actuating the stage and a capacitive position sensing
mechanism, capable of measuring displacements up to 4.5 µm with a resolution of 0.01 µm in both
X and Y direction is integrated to provide position feedback. Kim and Kim62 developed a single
crystal silicon (SCS) micro XY-stage. The fabrication involves DRIE and chemical mechanical
polishing (CMP). The fabricated actuator has a 2-D active driving region of 50 µm × 50 µm and the
sticking problem has been removed by deep etching of the sacrificial glass substrate. A maximum
travel range of 24 µm was obtained at about 20 V.
Figure 20: Schematic diagram of the micro XY-stage. 62 63
Advancing on their previous developed model, Kim et al.63 fabricated a micro XY-stage with a 5 ×
5 mm2 – area stage for application in nanometer-scale data storage. The square stage was
successfully released from the substrate using Micro-Channel Assisted Release Process (µCARP).
The displacement obtained was 36(±18) µm at 0.1 Hz and 96(±48)µm at 140 HZ for 12 Vp-p-quasi-
static triangular wave with 7.5 V-dc bias. The positioning range was 36 µm × 36 µm and the first
resonance frequency was 164 HZ with very little mechanical interference. 3-D stages were
designed and fabricated by Ando71 which were driven by electrostatic comb actuators. The stage
consists of travelling tables, suspensions and comb actuators and fabricated using inductively
coupled plasma etching (ICP), focused ion beam (FIB) and wet-etching. The maximum lateral
displacement obtained was 1 µm in the x-direction and 0.13 µm in the y-direction when the driving
voltage was 97 V and 105 V respectively and the maximum vertical displacement was 0.4 µm when
the driving voltage was 90 V.
Figure 21: Components of the nanopositioner: (a) ATIMs, (b) bistable mechanism, (c) sliding couplers, (d) TIMs, (e)
ampliﬁer mechanism and (f) gratings.64
Figure 22: Two DOF MEMS nanopositioner (SEM image)65
Hubbard and Howell64 developed a nanopositioner actuated by thermal microactuators. The device
is surface micromachined in a two-layer polysilicon process using MUMPs and consists of two
stages for independent coarse and fine position control. The first stage travels 52 µm between two
discrete positions, and the second stage mounted on the first stage, moves continuously through an
additional 8 µm in the same direction as the first stage, extending the total range to 60 µm at current
of approx. 141-159 mA and power of 6.6-7.6 V or 0.93-1.21 W. An open loop control was used for
the control scheme. Bergna et al.65 presented the design, modelling, fabrication and measurement
characteristics of MEMS nanopositioners that combine parallel bi-lever flexure mechanism with a
bent beam thermal actuator. The flexure mechanism amplifies and guides the motion of the actuator
with high precision and the thermal actuator provides the necessary force and displacement. The
authors applied an open-loop controller. The motion repeatability was found to be less than ±7 nm,
step sizes below 50 nm, the position resolution below 12 nm and the maximum displacement was
approximately 12 µm. Extending their previous work, researchers at the Intelligent Systems
Division at the National Institute of Standards and Technology (NIST), Gorman et al.66, Maryland
used three classical control approaches for controlling the MEMS nanopositioner including i) a
quasi-static nonlinear open-loop controller, ii) a nonlinear forward compensator, and iii)a nonlinear
PI controller. The authors used Laser Reflectance Microscope instead of SEM for achieving
position measurements with nanometer resolution.
Singh et al.67 at the Institute of Microelectronics, Singapore designed, developed and demonstrated
a single wafer process with a unique integration of bulk and surface micromachining process steps
(including KOH bulk micromachining, DRIE high aspect ratio micromachining and
Electrochemical etching [ECE]) for electrostatic microactuator for integration in 3-D micromirror
device. Bottom electrode, flexure springs and micromirror plate were fabricated in single crystal
silicon substrate layer, while the top electrode plate and bending springs were realized in
polycrystalline silicon layers. An angular deflection of 2° max was achieved. A micromachined XY
stage consisting of eight “L” shaped spider-leg stage-suspension springs and rotational comb-drive
actuators for two-dimensional microlens scanner was proposed by Kwon et al.68. The fabrication
was a combination of RIE and photolithography on a SOI wafer. The pitch of the microstage matrix
is 2mm, the stages consisting of five lenses with a 260 µm diameter and about 3 µm sag. The
fabricated stage was found to drive by lateral 55 µm in the X and Y directions independently with a
typical 40 V drive voltage. The fabricated XY stage was found to move more than 55 µm in the X,
Y directions independently at a driving voltage of 39.4 V.
Figure 23: (a) Schematics of the XY stage, (b) simpliﬁed model showing the stage motion to the right by using a pair
of rotational comb actuators on the right-hand side, and (c) diagonal motion by using the actuators on the top and right
sides.68 (d) Schematic view of the electrostatic comb-driven XYZ-stage with topological layer switch architecture.69
Takahashi et al.69 at the University of Tokyo reported a monolithic XYZ-stage with electrostatic
comb-mechanisms integrated in only two silicon layers and by three photolithography steps that
topologically switches the allocation of layer for electrical and elastic components. The XY-stage
moved in the X- and the Y-direction by 19 µm independently, and also in the diagonal direction and
the Z stage moved a maximum of 2.12 µm with an applied voltage of 200 V. Sun et al.70 fabricated
their nano-positioning micro XY-stage on a single-crystal-silicon using DRIE and anodic bonding
processes. The foot-print of the developed stage is 2000 × 2000 µm2 and is suspended at the center
of the wafer chip by four sets of folded beam, detecting beam and bending flexure composite
springs. The authors use a closed-loop controller and the displacement of the stage is detected using
position sensors (four piezoresistors fabricated by lateral diffusion process). The simulation and
analysis results showed a displacement of 13.5 µm and the effective driving force is 649.8 µN with
a driving voltage of 30V.
Figure 24: Solid model of the bulked micromachined 3-axis MEMS positioning stage.70
4. Hypothesis, Objectives and Questions to be answered
This research is fundamentally aimed to develop an automated cellular delivery and surgery device
using micro/nano fabrication technology. Thus it would involve the design and fabrication of a
micromachined XYZ stage-needle assembly that can deliver foreign materials like proteins, nucleic
acids, DNA among others into the cell that are of great importance for a variety of applications
including drug delivery and testing during neural recording into cells72, toxicology, neurobiology,
pharmacology and basic cell biology. It is important to quantify the distribution of behavior
amongst a population of individual cells to reach a more complete quantitative understanding of
cellular processes. This would lead to modulation of cell activities while monitoring the reaction of
the cell in real-time and also controlled differentiation or therapy of living cells. There are several
reasons which make the use of micro/nano fabricated devices and systems such breakthrough
technologies for cellomics and high-throughput screening (HTS):
• Various methods for manipulating large numbers and variety of cells simultaneously.
• Increasing interest in analysis of living single cells for studying the effects of drugs, external
stimuli on cell behavior etc.
• Easy integration of different analytical standard operations onto the microfabricated system.
• Alignment of the cell sizes with the microfabricated devices which range from 1-200 µm.
• Large electrical field strengths can be obtained with small voltages.
Microfabricated XY/XYZ-stages have been extensively studied in microoptic and data storage
systems. One of the main motivations of this research involving parallel manipulation of living
cells come from the early research done in a completely different area during the 1990s by Mamin
and Rugar73 at the IBM Almaden Research Center, where they pioneered the possibility of using an
AFM tip for read-back and writing of topographical features for the purposes of data storage. Later
in early 2002, IBM Zürich Research Lab74 invented the “millipede” technology (see figure 25)
which is a completely new approach for storing data at high speed and with an ultrahigh density. It
is based on a mechanical parallel x/y scanning of either the entire cantilever array chip of the
storage medium coupled with a feedback-controlled z-approaching and leveling scheme which
brings the entire cantilever array chip into contact with the storage medium. This tip medium is
maintained and controlled while x/y scanning is performed for write/read.
Figure 25: IBM's "Millipede" concept74
Some of the most obvious parameters that govern the application of these stages include maximum
stroke, access speed and precision. Unfortunately, it is not easy to realize a large displacement and a
high speed simultaneously, because the large stroke requires a flexible suspension that reduces
access speed due to the low natural frequency.
The second main motivation of this research stems from the recent identification and sequencing of
all the human genes which have brought unprecedented opportunities to understand molecular
mechanisms of development and disease75. Rapid genome-wide functional screens examining the
contribution of every gene to a biological process have been made possible76. For example, this
includes exposing cells to specifically designed dsRNA corresponding to a gene of interest, which
is then used by endogenous enzymes to recognize and destroy the corresponding messenger RNA
(mRNA), thus inactivating the gene function. This method is popularly known as RNA
interference, or RNAi77, which is a powerful tool for functional genomics research and potential
therapeutics78. Thus cell transfection techniques are powerful for in vivo and in vitro cellular and
molecular biology studies. As discussed above, there have been various efforts of developing novel
micro/nano-needle designs for such cell transfections.
Combining the above two motivational factors, this research seeks to develop a new kind of cellular
delivery and surgery device that can inject foreign materials into the cells at a high throughput rate
with enhanced flexibility and scalability. After extensive literature review, it was discovered that no
researcher has actually combined the advancements in microstage and micro/nanoneedle
technology for application in cell delivery and surgery. The usage of multiple probes with a specific
pitch as in the Millipede technology74 can result in large maximum stroke, because “switching to
the adjacent probe” is available when a stroke greater than the maximum is required. Thus, the
actual stroke of the device is given by “(displacement) × (number of microprobes)”. This means
that a large number of microprobes can compensate for the small stroke of an actuator and this
leads to large access area and a fast access time simultaneously. Another challenge is realizing
individual x/y feedback of the probes on a single microchip and then delivering foreign materials
into the cells based on the vision feedback and simultaneous z displacement. It should be noted that
even in a single cell line, cells highly vary in shapes and sizes and the position of the nucleus too.
For example, when delivering DNA into the cells is a prerogative, then the nucleus of the cell has to
be identified and then the delivery must take place. Apparently, the nucleus is not always at the
center of the cell and thus a precise control of independent movement by individual probes across
the three axes is essential to align themselves to compensate for the variable cell sizes and shapes.
Thus my main hypothesis lays on the fact that integrating these microprobes onto a 2-axis stage,
the simultaneous z displacement being provided by an external micromanipulator, can help attain
better control over the cellular delivery process. This can be realized when the individual probes on
the microchip can be actuated independently across X/Y axes and simultaneously across Z axis.
When this device is connected to the automated cellular injection system in a modified fashion, it
would help achieve a high throughput delivery rate along with precise control over automation of
Over the course of the next three years, I will try to answer the following questions:
1. How to fabricate an integrated system of arrays of micro XYZ stage-probe assembly on a single
2. How to sort and trap the cells onto a cell holding device? What would be its design? What
would be its fabrication procedure?
3. What would be the actuation principle (electrostatic/thermal) so that high displacement and
speed can be realized simultaneously without affecting the simplicity of design and fabrication?
4. How to design the open-loop control algorithm for precision motion control of the device?
What about the vision feedback?
5. How to integrate the automated cellular delivery and surgery device with the existing cell
injection experimental system? What kind of modifications need to be made for such
Thus, this research aims to achieve the following objectives, by answering the above questions:
1. Develop an integrated assembly structure of micro XYZ stage with individual probes on a
2. Develop a simple cell holding device that can sort and trap the cells.
3. Develop the control scheme that can provide nanometer resolution to the assembly. Currently
the proposed actuation method is electrostatic. My research would also analyze the feasibility of
thermal actuation for the device.
4. Develop the associated vision algorithm for identifying the cell sizes and the corresponding
nucleus, to obtain visual feedback for the controller to align the individual probes in x/y/z axes.
5. Integrate the automated device with the existing experimental system after suitable
6. Study the delivery of foreign materials into the cells and record the corresponding data related
to the insertion force, fracture force, success rate of delivery, survival rate of the cells, cell
trapping success rate and viability, resolution, repeatability, hysteresis and drift of the
microstage among others.
7. Perform statistical analysis of the data whenever essential.
5. Research Framework and Methodology
A series of three studies is proposed to investigate the delivery of foreign materials into the cells.
Specific aims and anticipated ways to achieve them are detailed below.
Aim 1: Design and fabricate 2-axis microstage-needle assembly device along with a custom
made cell sorting and trapping device.
Figure 26: Optical microscopic images of the microXY stage under operation68
Aim 1 proposes to develop a highly integrated system that combines arrays of micro/nano-needles
on arrays of X/Y micro-stage/scanner through monolithic microfabrication without any manual
assembly or alignment for cellular delivery and surgery. Such a compact system would involve
electrostatic comb-drive microstage that would enable both a large in-plane displacement and out-
of-plane actuation. The current scheme of actuation would be electrostatic actuation using parallel
plate capacitors. Nevertheless, this research would also investigate the use of rotational comb-drive
actuators for rotational x/y motion and thermal actuation. Two such rotational in-plane motions for
microoptics have been realized before by Kwon et al.68 and Singh et al.67.
Figure 27: SEM and optical micrographs of the rotational microstage (left) top view SEM (right) tilted view SEM67
Electrostatic actuation has been selected as our immediate actuation mechanism due to easier
selectivity of electrode materials, good repeatability in electromechanical positioning, smaller size,
low power consumption, higher switching speed and ease of integration with control electronics.
Nevertheless, there are several issues including sticking/charging, low force generation capability
and high voltage requirement. The z-axis movement would be provided to the entire array of stage-
needle assembly on the single microchip by an external micromanipulator. An immediate design is
shown below which consists of parallel plate electrodes, restraining beams that connects the
actuators to the stage, folded beam suspension springs to suspend the large number of comb
electrodes per direction, supporting beams and anchor to give support to the comb-drive electrodes,
electrical connections and the central stage on which there are arrays of micro/nano-solid needles
that are pyramid in structure. Once (a) arrays of needles on a single stage can be successfully
fabricated following detailed design criteria, the next immediate step would be to advance this
fabrication and design for (b) arrays of individual stage-needle assembly on a single microchip.
Figure 28: (a) Proposed design of parallel 5 x5 arrays of microstage-micro/nano-needle assembly (b) Exploded view of
an individual 2-axis microstage-micro/nano-needle structure of (a)
It is to be noted that every individual structure as part of the assembly is positioned orthogonal to
each other. This helps in reducing the X/Y axes cross talk. Some of the design criteria that has to be
taken into account to provide as much decoupling motion as possible includes the lateral and
vertical spring stiffness, lateral and vertical restraining beam stiffness among others. For e.g. the
stiffness of the folded beam suspension springs can be increased by increasing the number of folds.
The simulation of in-plane and out-of-plane displacement and x/y axes cross-talk would be done
using Finite Element Modeling software, e.g. ANSYS. In the above proposed designs, the inner and
outer fingers of the folded beam suspension spring are restrained. Modal analysis would also be
performed to confirm the vibration characteristics, such as resonant frequency and modal shape.
Following simulation and obtaining an optimized design of the assembly, the next step would
involve fabrication. The fabrication of the two of the early and major works in integrating
cantilever with microstage has been discussed briefly below.
Indermuhle and Rooij58 developed a micro xy stage with an integrated tip for atomic force sensing.
After having been oxidized and prestructured by photolithography, buffered hydrofluoric acid
(BHF) and reactive ion etching (RE), two 100 highly doped ( 0.05 Ωcm resistivity) silicon wafers
are glued together with aligned silicon fusion bonding (1 to 3). Then they are etched in potassium
hydroxide (KOH) until the upper wafer is about 70 µm thick. At the same time, via holes are etched
through the lower wafer (4). Wafers are then reoxidized and square caps of 130 x 130 µm are
patterned with photolithography and BHF etching; a further KOH etching is then performed to form
pyramidal tips (5) and to reduce the upper wafer to a 5 to 10 µm thick membrane. This membrane
and the one remaining in the via holes are pierced by dry etching to release the structures (6)58.
Figure 29: (Upper) Fabrication of the micropositioner with integrated large tip micromachined in monocrystalline
silicon. (Lower) XY microstage with a large tip58
Another prominent work as discussed above was at the IBM Zurich Research Lab Lutwyche et al.79
(figures 30 and 31). The following design is a 5 x 5 2-D AFM cantilever array for terabit storage
device. The starting material is a 100-mm silicon-on-insulator SOI wafer with a 15-µm-thick,
slightly n-doped silicon membrane, a 1-µm-thick silicon dioxide intermediate layer, and a 525-µm-
thick base. A 1-µm layer of thermal oxide is grown on both sides of the wafer and patterned on the
topside using a CHF3/O2 reactive ion etch RIE to form the mask for etching the tip and the
alignment marks, as in figure 29 (1). The silicon tips are made by an isotropic SF6/Ar RIE 6
process. This leaves the wafer as in figure 29 (2). The tips have been sharpened by thermal
oxidation and buffered hydrofluoric (BHF) acid etch or in potassium hydroxide solution. Next, the
piezoresistors and tip heaters are defined by lithography and by local p+ boron implantation at 80
keV with a dose of 1 x 1015 ions/cm2, figure 29 (3). A 30-min annealing at 1050 °C is used to
activate the dopants. A 250-nm-thick Si3N4 insulation layer is added to the wafer by plasma-
enhanced chemical vapor deposition and subsequently patterned. The contact holes to the silicon
are made using BHF, as in figure 29 (4).
Metal wiring for the piezoresistors and tip heaters is made by evaporating and wet-etching a 300-
nm aluminum layer, figure 29 (5). A 30-min annealing step at 450 °C is used to alloy the contacts.
A silicon dry-etch step using DRIE is used to pattern the cantilevers. To protect the tips during this
dry etch process, an 8-µm-thick photoresist layer is patterned and transferred into silicon. Figure 29
(6) shows the wafer at this point. Subsequent steps are performed on the back of the wafer. To
provide a stable etch mask for the backside wafer through-etching, a 12-µm-thick photoresist layer
is spun on the backside of the wafer. Double-side alignment lithography is used to pattern the
backside oxide in BHF, which is then used as the mask in another DRIE step.
During this process, the front side of the wafer is protected by photoresist. Stripping this resist is
the last stage of the process, so it protects the fragile cantilevers until they become free standing.
The intermediate SOI layer is used as etch stop, making it possible to achieve a homogeneous etch
depth over the entire 100 mm wafer. Figure 29 shows the wafer at this stage. The 5-µm-thick
silicon membrane on the topside of the wafer provides a stable compensation for the internal stress
of the SiO2 interface layer. The SiO2 is then removed in BHF and the silicon membrane is etched
from the backside until the silicon is etched through. Finally the resist is stripped away, leaving the
structure as in figure 29.
Figure 30: Silicon micromachining steps for cantilevers with tips and resistors. (a) Pattern oxide for tip, (b) etch tip, (c)
pattern piezoresistor, (d) pattern silicon nitride, (e) pattern aluminum, and (f) pattern cantilever. Backside silicon
micromachining steps for suspending cantilevers. (a) Deep trench etch through wafer, (b) cantilever release79.
Figure 31: SEM image of 5 x 5 2D cantilever array chip79
The design and fabrication of a microfluidic cell sorting and trapping device is also a prerogative.
Nevertheless, since the fundamental focus of this research is to automate the cellular delivery
technique and develop a micromanipulator as discussed above, the focus would be on a rather
It is proposed that the device would be fabricated out of glass. Liu and Sun80 used PDMS as a
sandwich between two glass layers for forming a vacuum chamber. Nevertheless, in our case, the
negative pressure would only be applied for sorting and trapping the cells to the vicinity of the
microwalls. Thus it is a matter of investigation if PDMS would actually be used or not and if it is
used, then for what purpose. The cells would be finally trapped using surface chemistry, for
example coating with a thin gold layer and the corresponding antibodies on the gold layer can
immobilize the cells81, or, the wall and well can be coated with cellular adhesion molecules, for e.g.
There are, nonetheless, several design issues that need to be addressed. The foremost being the
design has to be such that the electrical or thermal connections on the 2-axis stage-needle assembly
device does not come in contact with the conductive cellular medium which might damage the
device. A possible solution is to fabricate the needle with high aspect ratio and simultaneously
constrain the dimensions of the cell holding device such that the amount of cell medium is just
enough for cell survival and yet not too much so that it does not come in contact with the
electrical/thermal contacts. In addition the microwall must be wide enough for a cell, but not too
wide enough to house more than one cell; the microwell must be deep enough to protect the cell
from being washed away by flow during rinsing, but not so deep that more than one cell may be
trapped on top of one another; the flow of the cell culture medium, as the microarray is a squared
structure, the microwalls situated at the diagonal ends of the port have a less probability of
capturing cells than the ones far from the diagonal corners; the total time taken for immobilization
of the cells, among others. Extra untapped cells can be removed either by using a transfer pipette, or
by rinsing. Studies have to be made to study the effect of diameter and depth of the microwells on
cell trapping success rate, time of immobilization etc. Several other factors might also be
investigated, for example, the fluid velocity and shear stress simulation and modeling to determine
shear stress conditions for trapped cells to compare to physiologically relevant shear stresses.
Figure 32: Seeding of cells in PDMS microwells. (a-e) Schematics of the fabrication and seeding procedure: (a) PDMS
prepolymer is poured on to a photolithographically patterned master; (b) after curing, PDMS is peeled off and
microwell devices cut out (cut location indicated with dotted lines); (c) a PDMS microwell device is placed in a P35
Petri dish and PDMS is added to fill in the rest of the dish;(d) a cell suspension is allowed to settle on to the microwells;
(e) cells remain in the microwells after rinsing the excess cells from the top surface; (f) nine representative pictures of
RBL-1 cells in microwells for all the combinations of three different diameters (range D = 20-40 µm) and three
different depths (range H = 16-27 µm). There is an increase in the total number of trapped single cells as microwells
gets deeper (left to right) and narrower (top to bottom); wide microwells can accommodate multiple cells, but the cells
also tend to be more easily dislodged from the bottom of the microwells during the rinsing step. Scalebar, 100 µm.82
The fabrication can be done using soft-lithography technology and replicate molding. One such
similar fabrication was conducted by Rettig and Folch82. First the master molds were fabricated
using photolithographic microfabrication. Silicon wafers used as substrates for fabricating the
master molds were first baked to remove adsorbed moisture. Immediately after, negative
photoresist SU-8 was spread at ~550 rpm for 10 s and then spun for 30 s at 1300, 1700 or 2500 rpm
to yield feature heights of H = 25, 20, or 15 µm. Immediately after spinning, each wafer was placed
on a hot plate to evaporate the solvent and then they are ramped and exposed to ultraviolet doses on
a contact aligner using a dark-field mask. They were again placed on a hot plate, ramped, held and
cooled to selectively cross-link the exposed areas of the photoresist layer. Next the substrates were
immersed in SU-8 developer for 3-4 min each to remove non-exposed portions of the SU-8.
Masters were then exposed to a vapor of (tridecafluoro-1,1,2,2-tetrahydrooctyl)-1-trichlorosilane in
moderate vacuum for 1 h which renders the exposed silicon surface very inert, which facilitates the
release of the PDMS mold after curing. This was then followed for replica-molding of masters in
PDMS as shown in Figure 33. Nevertheless, it is a matter of investigation regarding the insulation
of the device when it is inside the cell culture medium, due to the electrostatic actuation involved.
Aim 2: Develop a control algorithm for actuating the 2-axis device with nanometer precision.
Correspondingly, develop a vision algorithm for vision feedback to the controller.
Aim 2 proposes to develop a control algorithm either in LabVIEW or MATLAB for the precision
control of the 2-axis MEMS assembly. Apparently, open-loop control is the most common
approach for MEMS because a) it does not require sensor feedback, simplifying the design, greater
scalability and eliminating the need for on-chip signal conditioning; and b) can be implemented on
many devices simultaneously without the need for high performance control electronics66. In
addition, our device would use a vision feedback to identify the location of the cells or the internal
organelles within the cell, for example, in this case, the nucleus and then scan the X and Y positions
accordingly, by giving the feedback to the control algorithm.
The relationship between input voltage and the resulting displacement for electrostatic actuators
would be investigated to find if it is linear or non-linear. There are a number of methods for
implementing the controller into our device, based on either a static or dynamic model of the
system. In two such related works, Gorman et al.66 and Sun et al.61 have found the nonlinear
inversion control scheme to provide the best results in terms of increased bandwidth, tracking
accuracy, robustness and control effort. Gorman et al.66 used a nonlinear PI control while Sun et
al.61 used nonlinear PID control coupled with linear system controller design.
Figure 33: Nonlinear model inversion position tracking61
Nevertheless, for the control algorithm to actuate the device, there has to be visual feedback to the
controller. Thus, a simple visual algorithm will be developed either in LabVIEW or MATLAB that
would perform three basic functions: a) automated scanning of cells b) automated alignment of the
stages with needles to align them on top of individual cells c) automated cellular delivery of the
foreign material. Thus, the control and vision algorithm would work in conjunction with each other.
Each of these functions has been described in brief.
• Automated scanning of cells: The very first step in pattern recognition will be the
computerized scanning of the microarrays in the cell sorting and trapping device and detecting
the individual cells. If DNA is to be injected, the nucleus has to be identified. Zappe et al.83 at
the Stanford University developed an automated MEMS injector for mass-injection of
Drosophila embryo. Their vision user-interface was named Stanford Automated Drosophila
Embryo Injection System. The image was first converted to a binary black-and-white picture
and then digital filter was applied to identify continuous dark areas that are too small or too big
to be embryos. The remaining continuous dark areas are considered images of embryos and
statistical data such as the total area and the center of this area are recorded. A corresponding
image with identified embryos is displayed in the right frame of the LabVIEW user interface.
Whenever an embryo covers a relatively small area and is located on the edge of one frame, it is
likely that a significant portion of it appears in a second frame as well. In such cases, all
collected embryo positions are analyzed and whenever a doubly detected embryo is found, one
set of coordinates is discarded to avoid double injection of embryos. Scanning of the 7 x 7 mm2
area takes 2 minutes.
Figure 34: Stanford Automated Drosophila Embryo Injection System83
In another such work, Wang et al.16 at the University of Toronto developed a microrobotic
system for fully automated zebrafish embryo injection. Their vision system was named as
UToronto Autonomous Cell Injection System. In recognizing the embryo structures, the
cytoplasm center was chosen as the deposition destination. However, the recognition algorithm
allows for choosing a different destination, for example, closer to the yolk/cell interface to
facilitate the diffusion of injected molecules into the cell portion. The recognition of detailed
embryo structures takes 45 ms on the host computer. Pre-processing is conducted to obtain de-
noised binary images. An image is first convolved with a low-pass Gaussian filter for noise
suppression. The gray-level image is then binarized to a black-white image using an adaptive
thresholding method, in which a local threshold for each pixel is set to be the mean value of its
local neighbors. The binary image is eroded to remove small areas that represent spurious
features and then, dilated to connect broken segments that originally belong to one object. Of
the connected objects in the binary image, the one with the maximum area is recognized as the
chorion (enclosed by its minimum enclosing circle). The second largest object in the image is
the cytoplasm, the boundary of which is represented by a chain code contour. The boundary of
the cytoplasm is often not fully connected; however, a fully closed contour is important for the
recognition of detailed cytoplasm structures including the yolk, the cell portion, and the yolk-
cell interface. Thus, a convex hull of the contour is constructed and used as initial positions for
subsequent snake tracking. Snakes, or active contours, are often used to locate object
boundaries and track deformable objects. They are energy minimizing splines influenced by
external constraint forces and image forces that guide snake points towards features such as
lines and edges. The centroid of the contour, O is recognized as the cytoplasm center. The
switching point, S is then determined as the intersect point of the minimum enclosing circle and
the horizontal line passing through the cytoplasm center. In order to distinguish the yolk from
the cell portion to provide the flexibility for choosing a desired destination, the cytoplasm
contour after snake tracking is fitted into an ellipse using a least squares method, and
intercepted into two parts by the minor axis of the fitted ellipse. Based on the fact that the cell
portion always has greater convex deficiency, the cell and yolk portions are distinguished. For
details on the image processing, the readers may refer to the article by Wang et al.84.
Figure 35: UToronto Autonomous Cell Injection System16
It is a matter of interest to develop a similar visual user-interface for vision feedback.
• Automated alignment of the stages with needles to align them on top of individual cell:
Based on this visual feedback to the control algorithm, the stage-needle assembly has to be
aligned with the cells trapped into individual microwells in the cell holding device. After the
initial alignment, it is possible to automatically place the tip of the needle at a defined distance
above the surface of the cell holding device for any given x/y position of the embryo x/y stage.
The cell holding movement would be manually controlled which is attached to a
micromanipulator, so that the reference objects appear at predefined positions on the screens
and the corresponding stage positions can be recorded. Albeit, manual involvement can be
eliminated, nonetheless, time constraints might hinder achieving multiple research objectives in
a span of 3 years.
• Automated cellular delivery of the foreign material: This will be discussed in the next aim
i.e. Aim #3.
Aim 3: Design and perform the cellular delivery experiment, recording and collecting the
corresponding data. Perform statistical analysis of the data to justify the feasibility of the
Aim 3 proposes to design and perform the experiments related to cell delivery and surgery. It is a
matter of further research regarding how to deliver particular molecules into the cell or specific
organelles inside the cell, for example, nucleus. On a broad scale, the components of the experiment
would typically include two micromanipulators, one for coarse positioning of the cellular delivery
device to the vicinity of the cell holding device, the other for bringing the cell holding device under
the focus of the optical microscope, a negative pressure source for giving the vacuum pressure to
the cell holding device, the 2-axis stage-needle assembly, an optical microscope fitted with a CCD
camera, a vision and control system for visualization, feedback and control of the process, a
computer system for computational purpose and an environment control system to maintain cell
cultivation conditions, such as temperature, pH, O2 and humidity. It is to be later decided if the
normal optical microscope would be replaced by an inverted microscope to incorporate enough
space to perform parallel single-cell manipulation and to install additional components eventually
needed for the experiments including environment control. Please refer to the block diagram below
for the anticipated experimental setup.
One of the premium advantages of our cellular delivery system is that it doesn’t use any
microfluidics to deliver foreign substances into the cell, rather it uses the chemistry to perform the
delivery process. For example, while delivering DNA into the cell nucleus, the tips can be coated
with plasmid DNA encoding by virtue of the surface adsorption force85. The tips can also be loaded
with fluorescent substances, for example modification of the tips by fluorescein isothiocynate
(FITC)56 or protein coated Quantum Dots46. Chen et al.46 reported the development of a nanoscale
cell injection system, nanoinjector, that uses carbon nanotubes attached to an Atomic Force
Microscope (AFM) tip, functionalized with a cargo via a disulphide linkage for delivery of cargo
into live human cells, for example, protein coated QDots in this case. The release of the cargo took
place following the reductive cleavage of the disulphide bonds within the cells’ interior, cytosol,
after which the nanoneedle was retracted by AFM control. The pyramidal needle tips can be
sharpened to nanometer diameter using various methods, for example focused ion beam (FIB)
etching56, stabilizing the tips by applying a polymer coating followed by laser ablation to expose a
small submicron length at the active injection tip site86, fortifying the needles by applying a thin
layer of carbon among others87 etc.
Figure 36: Preliminary Experimental Setup
Nevertheless, it is a matter of investigation regarding the several possible failure modes of the
microneedles including buckling, shear and bending. Some of the other parameters that has to be
taken into consideration based upon geometry of the needles, include the force required for
insertion into the cells, fracture force, and other factors related to the process such as success rate of
delivery of the foreign substances into the cell based on fluorescence, survival rate of the cells, cell
trapping success rate and the time for immobilization and the viability of such a process as
described above. The feasibility of the microstages can be gauged by measuring the parameters:
resolution (the smallest step that can distinguished in the motion of the), repeatability (random error
observed during a series of attempts to move to the same position from the same stage direction),
hysteresis (error involved in approaching a position from opposite directions) and drift (measure of
the stability of the stage over time).
Finally, an anticipated cellular delivery process has been briefly described below:
• The cell culture medium containing the cells is first delivered onto the cell holding device
through the individual perfusion inlets using a sterile syringe. The vacuum pressure is
subsequently applied to the device to sort and trap the cells, and then due to the surface
chemistry of the device, the cells fix to the individual microwells. It is to be noted that since the
negative pressure applied would suck a substantial amount of the cell culture medium, thus the
rate of flow of the medium has to be controlled.
• The micromanipulator holding the cell holding device is then controlled to bring it under the
focus of the microscope.
• The CCD camera then takes the cellular arrays’ picture and sends it to the computer for
processing. The cells are identified, if their specific internal organelles are to be identified such
as nucleus or cytoplasm, it is done accordingly and this result is sent to the controller.
• The micromanipulator holding the 2-axis stage-needle assembly device is then actuated to bring
the 2-axis device to the vicinity, positioning it just above the cell holding device. The relative
distance is a matter of investigation.
• The controller then sends this feedback to the device and the individual stage-needle assembly
is positioned in x/y according to the position of the cells, to align them together.
• The device is then actuated in the z-direction using the external micromanipulator to penetrate
the cell membrane and make the delivery process and then retract back at the same velocity. All
the cells in the microarray are injected simultaneously.
• The next microarray is then brought into the focus of the microscope and step #6 is repeated.
This is continued until all the cells in the microarrays have been injected.
6. Gantt Chart
7.1. Publication Plan
Here is a list of conferences that are of importance to this research. The results from the research
are anticipated to be sent to IEEE EMBS 2011, IEEE MEMS 2012 and IEEE MEMS 2013. An
anticipated journal publication plan follows this.
Year Conferences Submission Date
May 9‐13, 2011 IEEE ICRA, Shanghai (China) September 15, 2010
August 25‐27, 2011 IEEE CASE, Trieste (Italy) February 11, 2011
August 30‐September 04, 2011 IEEE EMBS, Boston (USA) February 09, 2011
September/October 2011 IEEE IROS, San Francisco (USA) March, 2011
January 2012 IEEE MEMS September, 2011
September/October 2012 IEEE IROS, Portugal March, 2012
May, 2012 IEEE ICRA, Minneapolis (USA) September, 2011
August 20‐24, 2012 IEEE CASE, Seoul (South Korea) February, 2012
August 29‐September 02, 2012 IEEE EMBS, San Diego (USA) February, 2012
January 2013 IEEE MEMS September 2012
• A review paper titled "MEMS technology for cellular delivery and surgery" is anticipated to be
sent to either of the two journals for review by December 2010 (a) Annual Review of
Biomedical Engineering or, (b) Current Opinions in Biotechnology.
• A paper with a possible title "Microfabricated XY stage-needle assembly for cellular delivery
and surgery" is anticipated to be sent for review to IEEE/ASME Journal of MEMS by June
• A paper with a possible title "Automated Cellular Delivery of HeLa Human Carcinoma Cells"
is anticipated to be sent for review to IEEE Transactions on Biomedical Engineering by
• Any further journal publication would depend on the time constraints and quality of work
available. It should also be noted that abridged versions of the journal papers may be sent to the
conferences as discussed above.
7.2. Project Supervisory Team
Senior Supervisor: Wenhui Wang (Mechanical Engineering)
Co-Supervisors: Geoffrey Chase (Mechanical/Bio-engineering) and Richard Blaikie (Electrical
Associate Supervisor: Drusilla Mason (Biology)
Additional Supervisory Committee Member: Yu Sun (University of Toronto) and Xinyu Liu
7.3. Equipments to be accessed
The equipments and facilities at The MacDiarmid Institute for Advanced Materials and
Nanotechnology based at the University of Canterbury will be accessed for the fabrication process.
• Electron Beam Lithography (Raith 150)
• Reactive Ion Etching (Oxford PlasmaLab 80Plus)
• Optical Microscopy (Olympus BX30 with digital image capture)
• Atomic Force Microscope (Digital Instruments Dimension 3100)
• Plasma Ashing
• Optical Lithography
• Thin Film Deposition
Other than this, equipments available in the Mechatronics Research Lab would be accessed for the
experimentations including micromanipulators, anti-vibration table, Optical Microscope, high
resolution digital camera and pressure source. The most critical element, the living cells would be
obtained from Dru Mason's lab in the School of Biological Sciences and the chemicals for the
surface chemistry would be obtained from Alison Downard's lab in the School of Chemistry.
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