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Volume 9 | Number 22 | 2009

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Miniaturisation for chemistry, physics, biology, & bioengineering

www.rsc.org/loc Volume 9 | Number 22 | 21 November 2009 | Pages 3165– 3312

Lab on a Chip
Featuring work performed in the Selvaganapathy Lab,
McMaster University, in Hamilton, Canada As featured in:
Volume 9 | Number 22 | 2009

Miniaturisation for chemistry, physics, biology, & bioengineering

www.rsc.org/loc Volume 9 | Number 22 | 21 November 2009 | Pages 3165– 3312

Lab on a Chip

Title: Microinjection in a microfluidic format using flexible and compliant

channels and electroosmotic dosage control
Pages 3165–3312

ISSN 1473-0197

Bashir Ren
3D cytometry Water toxicity testing

Chang Hosokawa
3D (bio)particle sorter Rapid SNP genotyping 1473-0197(2009)9:22;1-V

We present a PDMS-based device that enables microinjections inside

microchannels. An integrated microneedle is actuated by the compliant Selvaganapathy et al., Lab Chip,
deformation of the device and the reagent is pumped using electro- 2009, 9, 3202–3211.
osmotic flow. The device was successfully tested on zebrafish embryos.
Pages 3165–3312

www.rsc.org ISSN 1473-0197

Bashir Ren
Registered Charity Number 207890 Water toxicity testing
3D cytometry

Chang Hosokawa
3D (bio)particle sorter Rapid SNP genotyping 1473-0197(2009)9:22;1-V
 PAPER www.rsc.org/loc | Lab on a Chip

Microinjection in a microfluidic format using flexible and compliant

channels and electroosmotic dosage control†
Arash Noori,a P. Ravi Selvaganapathy*a and Joanna Wilsonb
Received 20th May 2009, Accepted 7th August 2009
First published as an Advance Article on the web 8th September 2009
DOI: 10.1039/b909961a
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We present a novel PDMS-based microinjection system in a microfluidic format with precise

electroosmotic dosage control. The device architecture is fully scalable and enables high-throughput
microinjections with integrated pre- and post-processing operations. The injection mechanism greatly
simplifies current methods as only a single degree of freedom is required for injections. The injections
are performed inside a fully enclosed channel by an integrated microneedle. Actuation of the needle is
achieved by the compliant deformation of the channel structure by an external actuator. Reagent
transport is achieved using electroosmotic flow (EOF) which provides non-pulsating flow and precise
electrical dosage control. The potentials used for injections were between 5 V–25 V. The electrical
properties and flow rates for the device were characterized for Zebrafish embryos and Rhodamine B
and Methylene blue in pH 10 buffer solution. We also propose a method to enable precise individual
dosing of embryos using direct electrical feedback. Additionally, we show that electrical feedback can
be used to verify the location of the needle inside the injection target. A preliminary viability study of
our device was conducted using Zebrafish (Danio rerio) embryos. The study involved the injection of
ultrapure water into the embryos in an E3 buffer, and resulted in embryos that showed normal
development at 48 hours.

Introduction endocytosis. Although this approach is relatively simple and safe,

The introduction of biomolecules into cells and embryos is an
integral part of drug development, genetic engineering and in-
vitro fertilization. It has been applied to create transgenic
mammals,1 improve drought tolerance in plants,2 and to repro-
gram skin cells into induced pluripotent stem cells (iPS) for
patient-specific stem cells.3 However, the efficient transfection of
materials still poses a problem, and a variety of techniques,
broadly classified as biological, chemical and mechanical are
actively being developed. Biological techniques employ geneti-
cally modified viruses (viral vectors) to introduce DNA into cells.
This method is highly efficient, as many viruses have evolved
complex mechanisms to overcome cellular barriers, but suffers
from high cytotoxicity, restricted targeting of specific cell types,
limited carrying capacity, production problems and high costs.4
Furthermore, there are concerns associated with the use of viral
vectors for applications in humans, such as neutralisation by
serum anti-bodies and immunogenic reactions.5 This has led to
the development of synthetic chemical vectors such as lipoplexes
(liposome complexes) to allow delivery of material into cells.
Prior to delivery, negatively charged DNA molecules are com-
plexed with uptake enhancing transfection reagents. These
complexes then bind to cells and are internalized, typically by
a
Department of Mechanical Engineering, McMaster University, Hamilton,
ON, Canada. E-mail: selvaga@mcmaster.ca; Fax: +1-905-572-7944; Tel:
+1-905-525-9140 x27435
b
Department of Biology, McMaster University, Hamilton, ON, Canada
† Electronic supplementary information (ESI) available: Schematic of
conventional capillary microinjection and images of laser and focused
ion beam (FIB) processed needle. See DOI: 10.1039/b909961a
efficiency remains poor due to low uptake across the plasma
membrane, inadequate release of DNA molecules and lack of
nuclear targeting.4
A major barrier to efficiency is the degradation caused by
enzymes within endosomes and lysosomes. Physical methods
such as the gene gun, electroporation and sonoporation over-
come some of these issues by forming transient openings in the
cell membrane to introduce molecules into the cytoplasm thus
avoiding endosomal and possibly lysomal degradation.6 This is
achieved either by particle bombardment or by applying electric
fields or ultrasound to induce changes in membrane perme-
ability. Although these methods increase transfection efficiency,
this increase is directly related to increased cytotoxicity.
Furthermore, they only provide limited, non-quantitative
reagent transfection. In addition, degradation by enzymes within
the cytoplasm remains a problem.6
One physical method that overcomes the aforementioned
problems is capillary microinjection. Transfection is achieved
through the direct insertion of a microinjection needle into the
cell using a microscope and precision micromanipulators (see
Supplementary Fig. 1†). Two capillary microinjection techniques
exist, differing in the mechanism used for reagent transport.
These are capillary pressure microinjection (CPM), which uses
pressure driven flow (PDF), and ionophoresis which employs
electrokinetic principles (electrophoresis) for reagent transport.
Of these two methods CPM is most widely used as it is the
simplest and most direct way to inject extracellular material in
a targeted fashion (cytoplasm or nucleus) without cell or reagent
restrictions.6 A wide range of substances, including naked DNA,
RNA, antibodies and nanoparticles have been injected into cells

3202 | Lab Chip, 2009, 9, 3202–3211 This journal is ª The Royal Society of Chemistry 2009
 with high transfection efficiency (up to 100%) and low cytotox-
icity.7 However, the use of PDF for reagent delivery limits the
efficacy of CPM. It has been shown that the volume delivered
into cells may vary by a factor of 5 or more, resulting in signif-
icant variability and low reproducibility of injections.8 The use of
PDF also imposes limits on the minimum injection needle size
because of the high pressures that are required for dosing.
Existing commercial injection systems operate in the order of
500 kPa which restricts the minimum tip diameter to 0.2–0.5 mm.
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This limitation on the tip diameter is significant as smaller tips
lead to increased viability as they reduce the damage inflicted
onto cells during injection.9 To enable the use of smaller needles,
non-pressure driven reagent delivery such as ionophoresis may
be employed. Ionophoresis involves the insertion of electrodes
into the injection needle and into the cell medium to generate an
electric field that induces electrophoresis. Ionophoresis over-
comes the limitations on tip size as it is independent of needle
geometry.10 However, reagent delivery is slow and is dependent
on the properties of the ions to be injected. Furthermore,
quantitization of the reagent delivered remains a challenge.11
These factors make ionophoretic reagent delivery impractical for
clinical studies.
In addition to the limitations associated with the reagent
delivery, capillary microinjection suffers from low throughput
and variability as it is an operator-mediated process. It requires
a trained operator to perform the injections, which allows only
for a few hundred transfections per experiment. This makes
capillary microinjection impractical for studies that require
statistically significant sample sizes. Furthermore, the success
rate of injections is largely dependent on the operator’s ability to
orient the needle and holding pipette in a three-dimensional
space. This results in variability and low reproducibility due to
human error. To address these issues, a number of semi and fully
automated microrobotic injection systems have been developed
to enable rapid injection of Drosphila12 and Zebrafish13 embryos.
However, these standalone systems are costly, lack integrat-
ability and cannot be scaled easily which makes them unsuitable
for studies that require large sample sizes, as in the field drug
discovery. This problem of integration and throughput can be
addressed by the application of microfluidic technology. Per-
forming cell injections in a microfluidic format allows for inte-
gration of pre and post-processing operations such as cell
culture, sorting, and viability testing. Integration also reduces
potential damage to the cell caused by changes in the cell envi-
ronment.14 Other advantages of microfluidics include greater
selectivity, reduced dead volume and automation.15 Microfluidic
technologies have been applied in a number of devices for
microinjection of cells.16–18 Of these devices, only the work of
Adamo and Jensen17 approaches true high-throughput injec-
tions. The device features a stationary injection needle where cells
are impinged on by hydrodynamic pressure. Although it is
a promising technology, the functionality of the device is limited
and it is not truly scalable. As the needle is stationary in the
device, it does not compensate for differences in cell sizes and
also loses location selectivity for the injection. Furthermore, their
device uses pressure driven flow for injections and therefore also
suffers from dosing and needle size restriction issues.
Consequently, as a result of the inadequacy of current
microinjection systems, the systems that are presently used
This journal is ª The Royal Society of Chemistry 2009
to achieve high-throughput employ primarily chemical or
electroporation-based transfection. Some examples include the
Amaxa Nucleofector, Cellectricon Cellaxess system and the
MaxCyte Platform system. However, these systems only
provide non-quantitative and non-specific reagent transport, and
transfection efficiency remains low for some cell types. These
systems lack the flexibility and features that capillary microin-
jection offers, such as targeted delivery without cell or reagent
restrictions and up to 100% transfection efficiency.
In this paper we present a novel microinjection and reagent
delivery system that is fully scalable and maintains the advantages
of capillary microinjection and obviates the limitations. Our
device enables fully integrated high-throughput injections by
employing a simplifed microinjection mechanism which exploits
the compliance and flexibility of PDMS (poly-dimethylsiloxane).19
The system architecture is fully scalable and enables true minia-
turization, massively parallel injections and automation. It over-
comes the limitations on the needle tip size, minimum reagent
volume, and obviates the need for specialized pumps by per-
forming reagent transport by electroosmosis.20 This provides
precise electrical dosage control with virtually unlimited resolu-
tion and also scales more favorably (r2) than pressure driven flow
(r4). Another advantage is that using electroosmotic flow for
reagent delivery is less invasive than pressure driven delivery. One
study involving the microinjection of N. tabacum cell lines using
pressure driven and electrokinetic (ionophoretic) flow has shown
significantly different and favorable expression for the latter, likely
due to the damage that is caused by the application of pressure.21
Therefore, we believe that the use of electroosmotic reagent
delivery may also result in more favorable and accurate results for
injections. The device working principle and characterization are
discussed in the following sections.
Working principle
Device layout
The device, shown in Fig. 1(a) and (b), consists of a flexible
PDMS substrate with two separate and fully enclosed channel
structures that are 2 mm wide and deep. One channel supplies the
injection targets and the other channel the reagent. A hollow
glass capillary (1 mm OD, 0.5 mm ID) is embedded into
a channel (1 mm wide, 1.5 mm deep) perpendicular to the target
supply channel and is used to immobilize the targets using
suction. A tapered microinjection needle (15 mm OD, 7.5 mm ID
tip) is embedded into a channel collinear to the suction capillary.
Glass capillaries (2 mm OD, 1.5 mm ID) are used as inlet and
outlet ports. The assembled PDMS substrate is bonded onto two
separate glass substrates, one which is fixed and one which can be
actuated linearly. The final device is loaded into a fixture, shown
in Fig. 1(b), which ensures precise actuation of the needle.
Loading and sorting of targets
The targets are initially placed into a Petri dish and are loaded
into the target supply channel through a tube that is connected to
the inlet port. The loading and sorting process are depicted in
Fig. 2. The tube is brought in close proximity to the target which
is then loaded by applying suction to a syringe that is connected
to the outlet port of the target channel. Once a target has been
Lab Chip, 2009, 9, 3202–3211 | 3203

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Fig. 1 (a) Schematic of the fully enclosed microinjection device with two
channel structures for the targets and the reagent. (b) PDMS device
loaded into a fixture which immoblizes the left substrate and ensures
planar and linear motion of the movable substrate.
Fig. 2 (a) Loading nozzle lowered to embryo. (b) Embryo is loaded by
suction. (c) After injection the embryo is transported to the viable or
waste syringe reservoir.
loaded, the tube is raised to prevent the loading of other targets,
while ensuring ininterrupted flow of the buffer. The advantage of
this approach is that the target remains in the original buffer
medium which reduces environmental shocks that can affect
viability.14 Loading can proceed in a continuous fashion with
predetermined spacing between each target. Once the injection is
completed the target is transported into the appropriate viable or
waste reservoir.
3204 | Lab Chip, 2009, 9, 3202–3211
Injection using channel compliance
The sequence for the injection process is depicted in Fig. 3 which
shows the open cross-section of the device. When a target has
been loaded into the device, it is transported to the injection site,
as shown in Fig. 3a. The target is then immobilized by applying
suction to the suction capillary, shown in Fig. 3b, using an
attached syringe. As the target is immobilized in alignment to the
injection needle, only a single degree of freedom (DOF) is
required for injection. The injection mechanism takes advantage
of the flexible nature of the PDMS substrate. This allows for the
actuation of the needle by the compliant deformation of the
target channel top and bottom walls, shown in Fig. 3c. This is
achieved by the selective reinforcement of the PDMS substrate by
two separate glass substrates, one which is fixed and one which
can be actuated linearly. All regions except for the bottom of the
target supply channel are supported by glass substrates. There-
fore, applying a linear displacement to the movable substrate
causes the actuation of the needle by the deformation of the
target supply channel. To ensure precise linear motion of the
injection needle, the device is loaded into a glass fixture, shown in
Fig. 1b, which constricts the motion of the movable substrate
along the longitudinal direction of the needle. This also prevents
possible damage to the targets by the lateral movement of the
injection needle. The linear displacement that is required for the
actuation of the movable substrate is applied using an adjacent
linear micropositioner. The described injection mechanism
greatly reduces the complexity and variability associated with
microinjections. Only one DOF is required to perform injections
which compares favorably to conventional microinjection which
involves up to 5 DOF for the suction capillary and the injection
needle. Furthermore, as the injection occurs in a planar format it
provides precise control over the microinjection location and
facilitates automation. The required actuation is presently
applied using a manual actuator but can easily be automated due
to the simplicity of the mechanism. The mechanism obviates the
need for trained operators and reduces the complexity and
variability associated with capillary microinjections.
Fig. 3 Open device cross section. (a) Target approaches injection site.
(b) Target is immobilized by suction capillary. (c) Needle is actuated by
the compliant deformation of the PDMS substrate by an external
microactuator. (d) Conduction path for the potential that is applied to
induce EOF.
This journal is ª The Royal Society of Chemistry 2009
 Pumping of the reagent using electroosmotic flow
Once a target has been immobilized and punctured by the
microinjection needle, the reagent transport is initiated. The
pumping of the reagent is achieved by electroosmotic flow.22
The EOF is induced by applying a potential to electrodes that are
embedded in the target and reagent supply channels. The elec-
trical schematic for injections is shown in Fig. 3d. The conduc-
tion path is through the reagent solution, across the injection
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needle, the injection target and the target medium. The advan-
tage of employing EOF for reagent transport is that it obviates
the need for specialized pumps and provides true scalability to
the injection needle. Unlike conventional pressure driven injec-
tions systems, it does not place limitations on the size of the
needle. Furthermore, EOF provides a non-pulsating steady plug-
like flow and thus allows for precise electrical dosage control with
sub-picoliter flow rates.
Detection of needle clogging or breakage
Another challenge in conventional CPM is the detection of
damaged needles. Early detection is crucial as clogged or
damaged needles may lead to variation of the reagent volume
that is delivered, or no delivery at all. This may lead to incon-
sistent results and low reproducibilty for studies involving
transfections. To improve reproducibility, an efficient system for
detection is required to allow for remedial actions, such as the
flushing or replacement of the needle, to take place. In our
device, these changes can be monitored using electrical feedback
from the embedded electrodes. By monitoring the current levels it
is possible to detect changes to the needle, as well as when the
needle makes contact with the target. Although this has been
shown previously,23 in this work we show that this method may
also be used to provide real-time information about the location
of the needle inside the target. This further improves location
selectivity and provides an added, non-visual feedback mecha-
nism for automation.
Fabrication
The device was fabricated using PDMS soft lithography.24 The
process started with the fabrication of a negative base mold,
shown in Fig. 4a. A 3D polymer printer (Dimension BST768)
with an acrylonitrile butadiene styrene (ABS) working material
was used for the fabrication of the mold. This was done as the
Fig. 4 (a) Mold fabricated by 3D polymer printer. (b) PDMS substrate
cast and peeled from mold. (c) Exploded view of the device. The PDMS
substrate is bonded onto the glass slides. The suction capillary, injection
needle, inlet/outlet tubes and the electrodes are bonded into the device.
The device is fully enclosed using a 150 mm thick PDMS membrane.
This journal is ª The Royal Society of Chemistry 2009
feature sizes required for the mold made it impractical to use
photolithography for fabrication. Furthermore, using the poly-
mer printer obviated the need for costly cleanroom access and
enabled true ‘rapid prototyping’ as the molds were fabricated in
a span of minutes rather than hours. Once the base mold fabri-
cation was completed, PDMS pre-polymer was cast into the
mold to obtain the final PDMS substrate. To provide the
required flexibility for the final device, a 1 : 30 volumetric mixture
of curing agent to base was used for the PDMS. Control over the
thickness of the cast was achieved by casting a pre-determined
volume into the mold. This resulted in a 0.5 mm bottom wall
thickness for the target and reagent channels. After casting the
pre-polymer solution, the mold was placed onto a level hotplate
and allowed to cure at 65  C for 4 hours. After curing was
completed, the PDMS substrate was peeled from the mold,
yielding a positive impression as shown in Fig. 4b. The peeled
PDMS substrate was then selectively bonded onto two separate
3  100 glass slides using PDMS pre-polymer as adhesive agent.
The PDMS substrate was bonded onto the glass slides such that
all regions except for the bottom of the target supply channel
were reinforced by the glass slides. This required a spacing of
2 mm between the glass slides to accomodate the target supply
channel width. To ensure accurate alignment, a fixture was used
to immobilize the glass slides in a parallel alignment and with
a 2 mm spacing. The PDMS substrate was then carefully placed
onto the glass slides and aligned under a microscope. Subse-
quently, it was placed onto a hotplate at 65  C for 10 minutes to
cure the PDMS pre-polymer adhesive. After the PDMS substrate
was fixed onto the glass slides, the suction capillary and injection
needle were assembled into the device. The PDMS substrate
features two colinear 1 mm-wide and 1.5 mm-deep channels into
which the suction capillary and injection needle were bonded.
One channel intersects the target supply channel and houses the
suction capillary, and the other connects the target supply and
reagent supply channels and houses the injection needle. This
provided the colinear allignment of the suction capillary and the
injection needle and a 0.5mm spacing from the top and the
bottom wall of the channels. The channels were designed to
provide a flush fit for the 1 mm OD/0.5 mm ID borosilicate glass
tube (Sutter Instruments) that was used for the suction capillary
and in the fabrication of the microinjection needle. The micro-
injection needle was fabricated using a Sutter P97 micropipette
puller. The microinjection needle was manually cleaved using
fine tweezers under a microscope. The tip size of the pulled needle
was measured from digital images using the dimensions of
a standard 1 mm glass capillary as reference. From these images
it was then estimated at what location to cleave the needle using
the tweezers in order to yield a 15 mm OD/7.5 mm ID tip diam-
eter. Subsequently, the tip diameter was measured again to
ensure it met the requirements. The suction capillary and the
injection needle were then assembled into the 1 mm wide chan-
nels and aligned under a microscope. The suction capillary was
aligned to be flush to the side walls of the target supply channel.
The needle tip was positioned to extend 1 mm into the target
supply channel. Proper alignment was achieved using measure-
ments from digital images and making the necessary adjustments
to the positioning of the needle. Once a satisfactory alignment
was obtained, the suction capillary and injection needle were
permanently sealed into the PDMS mold. This was achieved by
Lab Chip, 2009, 9, 3202–3211 | 3205
 depositing drops of PDMS pre-polymer into the 1 mm  1.5 mm
channels into which they were assembled.25 Care was taken to
prevent overflow and clogging of the components by the PDMS
pre-polymer. The device was then placed onto a hotplate at 65  C
for 5 minutes to allow the pre-polymer to cure.
Subsequently, the channel inlet and outlet ports for the device
were created using 2 cm long hollow glass capillaries with a 2 mm
OD and 1.5 mm ID. The tubes were bonded into holes that were
punched into the inlets and outlets of the channels and sealed
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using PDMS pre-polymer. The final components to be assembled
into the device were the electrodes. The electrodes are used to
apply the potentials required to induce the electroosmotic flow
through the injection needle. We used 30 gauge stainless steel
needles as electrodes. The electrodes were inserted into the target
and reagent channels from the side, taking care not to obstruct
them. They were then bonded into place using PDMS pre-
polymer. After all the components were fully assembled the
device was fully enclosed using a 150 mm thin PDMS membrane.
PDMS pre-polymer was deposited onto the assembled device
and the membrane was carefully rolled on. Care was taken to
prevent the trapping of air bubbles. The membrane was fabri-
cated by spinning a 1 : 30 crosslinker : base mixture of PDMS at
400 rpm for 60 s onto a silicon wafer. The silicon wafer was
coated with parylene to facilicate the peeling of the membrane.
After the membrane was deposited, the device was placed onto
a hotplate at 65  C for 20 minutes. The fabrication of the device
was completed by connecting Tygon tubing to the inlets, outlets
and the suction capillary, and connecting 100 mL syringes to the
outlet tubing.
The most time consuming part in the assembly of the device
was the cleaving and the alignment of the needle. Therefore, we
investigated other methods that could simplify and potentially
automate this step in the assembly process. The other methods
that were explored were the use of a femtosecond laser and
a focused ion beam machine. They were used to cleave the needle
to yield a specified tip diameter. We used a Spectra Physics
titanium-doped sapphire chirped-pulse amplifier (CPA) laser
system to cut the needle in-situ. The process required 5 passes
and 10 s to cut the needle at a 15 mm wide section. A translational
speed of 500 mm/s and 0.903 mW power level were used for this
process. The results are shown in Supplementary Fig. 2.† The
image in (a) shows the uncut needle, (b) and (c) show the needle
after 2 and 5 passes of the laser, respectively. Using the femto-
second laser allowed us to very rapidly cleave the needle at
a required location. One advantage of this technique is that the
processing can be done in-situ prior to the sealing of the device. It
can also be used to control the length by which the needle extends
into the target channel. These features make the femtosecond
laser an attractive option for the processing of the needle in
a production environment with high volumes. For the present
device the needle was cleaved manually using the process
described earlier, as the laser is only economical for large
production volumes.
Another method that was explored for the cutting of the needle
was the use of a Focused Ion Beam (FIB) system (LEO/Zeiss
1540XB FIB/SEM CrossBeam). The FIB uses a beam of high-
energy gallium ions to mill and ablate an underlying material.
The FIB enabled us to cut, shape and sharpen the tip of an
injection needle. The results of the FIB sharpening process are
3206 | Lab Chip, 2009, 9, 3202–3211
shown in Supplementary Fig. 3.† Images (b)–(c) show the
gradual sharpening of the needle, and (d) shows a frontal view of
the sharpened needle. It can be seen that the width of the tip was
reduced from about 20 mm to 10 mm. The sharpening of the tip
could reduce the amount of damage that is caused by the needle
during injections. The FIB allowed us to fabricate highly
customized needle tips, which is not possible using conventional
micropipette fabrication techniques. Although this is promising,
there are also a number of limitations associated with the use of
the FIB. For instance, the cutting of the needle could not be
performed in-situ due to the limited size of the FIB/SEM loading
chamber. Furthermore, it required the samples to be made
conductive to prevent the charging of the sample. This was
achieved by sputter coating the sample with gold. For us this was
not feasible since making the entire device conductive would
have prevented the electroosmotic reagent transport across the
needle. To enable the use of the FIB for our device, the needle
would have to be sharpened and have the gold etched off prior to
assembly. This makes the assembly process increasingly complex.
Furthermore, cutting the needle with the FIB is a costly and time
consuming process which requires several hours. Therefore, the
FIB is presently impractical for application in our device fabri-
cation, and manual cleaving is the most appropriate method for
the fabrication of a small number of devices. The results pre-
sented in the following sections were obtained for a manually
cleaved microinjection needle.
Results and device characterization
The final device was used to inject Zebrafish embryos (Danio
rerio) with methylene blue, rhodamine B and ultrapure water
solutions. Zebrafish were selected as they are widely used as
model organisms for developmental studies, toxicology, genetic
engineering and drug discovery.26,27 The primary techniques that
are presently used to transfect Zebrafish embryos are manual
microinjection, which suffers from low throughput and vari-
ability, and chemical delivery agents such as the Endo-Porter,
which suffer from poor efficiency and limited carrying capacity.
Our device overcomes the limitations of these methods and offers
potential for high-throughput, efficiency and specificity. The
injection of a Zebrafish embryo using our device is shown in
Fig. 5. The first image shows the embryo immobilized by the
suction capillary. After immobilization, the embryo was punc-
tured by the needle by actuating the movable substrate. A
methylene blue solution was then pumped into the embryo by
applying a 25 V potential for 10 s to the embedded electrodes.
Upon completion of the reagent transport, the needle was
retracted and the embryo was dislodged from the suction capil-
lary by applying a positive pressure. The embryo was then
transported in the target channel to the final storage reservoir. In
this example, the process of actuating the needle and pumping
the reagent into the embryo took 15 s. The time required for the
injection is dependent on the actuation speed of the needle and
the reagent volume that is needed. Inserting and retracting the
needle from the embryo took 5 s and was achieved using
a manually-operated linear micropositioner. We expect that this
can be reduced to 1–2 s by using electric actuators. Therefore, the
rate-determining step for the injections is the time required for
the reagent transport. This is going to vary based on the
This journal is ª The Royal Society of Chemistry 2009
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Fig. 6 Relationship between the applied displacement and the displace-

Fig. 5 (a) Zebrafish embryo immobilized by suction capillary. (b) ment that is transferred to the needle.
Needle inserted into yolk sack. (c) Electroosmotic pumping of methylene
blue solution into the embryo by the application of 25 V for 10 s. (d)
Needle retracted from the embryo. Reducing the width of the material between the channels and the
edge of the device from 5 mm to 1 mm increased the displacement
transferred only from 83.8% to 84.2%. Reducing the length of the
properties of the embryo, the reagent and the reagent volume
compliant channel from 45 mm to 25 mm increased it to 83.9%.
that needs to be injected. Given the flow rates achieved in our
Changing thickness of the top membrane from 150 mm to 50 mm
system we expect this to be in the order of seconds.
increased it to 85.2%. These results show that the edge width and
The following sections discuss the characterization of the
the length of the channel have insignificant effects, and the top
device in greater detail. The results obtained for the needle
membrane only a small effect on the actuation of the needle. The
actuation and the electroosmotic reagent transport are pre-
bottom membrane thickness had the greatest effect on the
sented. A preliminary viability study involving the Zebrafish
percentage of the applied displacement that is transferred to
embryos is also discussed.
the needle. Reducing the bottom thickness from 0.5 mm to
0.1 mm increased the percentage of the applied displacement
transferred from 83.8% to 95.4%. Therefore, the channel bottom
Needle actuation using channel compliance
thickness is the most critical parameter and needs to be precisely
The actuation of the needle was characterized to ensure precise controlled to ensure repeatability. In our fabrication process this
control over the injection location. To achieve this, it was
necessary to determine how much of the displacement applied to
the movable substrate was transferred to the needle. This was
done by applying a known displacement to the movable substrate
and measuring the position of the needle relative to the suction
capillary using images that were taken. The measured experi-
mental values, depicted in Fig. 6, showed the effective needle
displacement as 83.8% of the displacement applied to the
movable glass substrate. This difference was caused by the
reaction forces induced by the application of the displacement.
Part of the applied displacement resulted in elastic strain in the
PDMS in the regions adjacent to the target channel. This reduced
the effective amount of the applied displacement that is trans-
ferred to the needle. The experimental results were compared to
results obtained from finite element simulations and were found
to correspond very well. The simulation results are shown in
Fig. 6. The conformance of the experimental and simulated
results confirmed the veracity of the finite element model. This
enabled us to conduct additional simulations to investigate the
relationship between the device geometry and the effective needle
actuation. The parameters that were investigated were the target
channel top and bottom wall, the length of the compliant target
channel and the lateral distance between the channel and the
device edge. The results obtained from the simulation of these Fig. 7 Simulation data for geometric parameter variations and their
geometric parameter variations are summarized in Fig. 7. effects on the applied displacement that is transferred to the needle.

This journal is ª The Royal Society of Chemistry 2009 Lab Chip, 2009, 9, 3202–3211 | 3207
 was achieved by casting a set volume of PDMS into the master
mold.
Electroosmotic injection
The characterization of the electroosmotic injection mechanism
was performed using rhodamine B (0.005 g/mL) and methylene
blue (0.04 g/mL) solutions. The buffer solution that was used was
a pH 10 carbonate buffer. This buffer solution was chosen as
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rhodamine B is electrically neutral in the range of pH 6–10.8.28
This electrically neutral marker allowed us to ensure that the flow
of the molecules through the needle was in fact due electroos-
mosis and not electrophosis. This provided us with a baseline to
investigate the behaviour of charged species. To investigate the
difference in behaviour between the electrically neutral and
charged species, measurements were obtained for methylene
blue, also in pH 10 buffer solution. Although the cells and
embryos to be injected would not be in a pH 10 solution, this
solution was used as it enabled us to study the relative difference
in the behaviour of the electroosmotic flow for charged and
neutral molecules. For actual transfection studies the device
would have to be characterized for the particular buffer/reagent/
cell combination under consideration. Presently, in order to
study the performance of the device, the flowrates and electrical
behaviour were characterized in the pH 10 buffer for a potential
range of 5 V–25 V. The required potentials were applied to the
embedded electrodes using a Keithley 2410 source/measure unit
interfaced with LabVIEW. The LabVIEW software enabled us
to record measurements at intervals of 0.1 s. The resulting
voltage–current data for the electroosmotic flow of rhodamine B
and methylene blue are shown in Fig. 8. The data was measured
while the device was in an un-injected state. The currents
measured at steady state are summarized in the inset graph. It
can be seen that the values obtained for the rhodamine B solution
were on average 19% greater than those obtained for the meth-
ylene blue solution. For the potential range of 5 V–25 V the
measured currents ranged from 0.12 mA–0.60 mA for the
rhodamine B, and 0.095 mA–0.53 mA for the methylene blue
solution. For both solutions the measurements were conducted
Fig. 8 Current–time response for methylene blue (MB) and rhodamine
B (R) solutions in un-injected state. Inset graph shows steady-state
currents.
3208 | Lab Chip, 2009, 9, 3202–3211
for a period of 10 s in order to better understand the current
response. It was found that the current behaviour for both
solutions was similar and stable. However, there was a slight
transient response for the rhodamine B flow, but this stabilized
after about 1 s. The transient behaviour can be seen in the first 1 s
of the data shown in Fig. 8. The transient response represents the
time required for the momentum of the double layer to be
transferred to the bulk of the fluid and for the flow to reach
steady state. The methylene blue solution exhibited a smaller
transient than the rhodamine B solution. This can be explained in
part by the greater conductivity of the solution which allowed the
electroosmotic flow to develop more rapidly. One factor that
could have caused transient behaviour for both solutions is the
non-uniform, tapered shape of the microinjection needle. This is
because as the inner diameter increases, the liquid in that section
requires longer to reach steady state. This problem could be
addressed in the future by using injection needles with uniform
inner diameters. Overall, however, the observed transient
behaviour was very small and is therefore not expected to
significantly impact the volume of reagent that is delivered.
The results discussed above were obtained for the device in an
uninjected state. The current behaviour of the device during
injections into Zebrafish embryos was also characterized. These
experiments were performed using the rhodamine B solution.
The embryos were injected for 10 s at potentials ranging from
5 V–25 V and the current response was recorded. The data
plotted in Fig. 9 is the difference, D, between the currents in
uninjected and injected state. The current values at steady state
results are summarized in the inset graph. The transient response
of the rhodamine solution during the first 1 s of potential
application can also be clearly seen. Overall, the data exhibited
consistent and predictable behaviour, however, it became slightly
unstable at potentials greater than 20 V. Therefore, using
potentials lower than 20 V may provide more accurate results in
the case of the electrically neutral rhodamine B solution. For
other solutions the optimal range will vary depending on their
behaviour during electroosmosis. For the rhodamine B solution,
the current values that were obtained during injections ranged
from 0.08 mA–0.47 mA. These values were on average 26% lower
than for the device in the uninjected state, due to the added
Fig. 9 Difference in current–time response in injected state and un-
injected state for rhodamine. Inset graph shows steady-state currents.
This journal is ª The Royal Society of Chemistry 2009
 resistance of the embryo. This offset is shown in the inset graph
in Fig. 9. The current measurements were used to deduce an
added resistance of 15 MU during injection into the embryo.
The associated joule heating during injections was calculated to
be between 0.4–11.8 mW. It was also found that almost 70% of
the potential drop occured across the microinjection needle.
Therefore during injections the embryo was only exposed to 30%
of the applied potential, which would have been 7.5 V at 25 V.
Assuming that the injection occured into the center of the
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embryo this would have exposed the embryo to a maximum
electric field of 150 V/cm at 25 V. This is significantly lower than
the electric field strengths of up to 3500 V/cm that are used in the
electroporation of Zebrafish embryos.29 In addition, as opposed
to electroporation, our method minimizes the damage to the
embryo by localizing the joule heating caused by the electric field
during injections. Therefore, it can be expected that the viability
rates obtained with our device will exceed those obtained by
using electroporation.
Another advantage of using an electrical system for our device
was that it allowed us to monitor the location of the needle
during injections. During the characterization of the device it was
found that the current response during injections varied signifi-
cantly from the one in the uninjected state. We observed distinct
differences between the shape and magnitude of the response
depending on the state of the device. Further measurements were
obtained for the needle in the uninjected state and when it was
injected into the chorion and the yolk sack of the embryo. The
potential used was 10 V. The current response is shown in
Fig. 10. The images to the right denote the corresponding loca-
tion of the needle in the embryo. It can be seen that inserting the
needle into the chorion caused a significant downward shift of
the current from around 0.26 mA to 0.20 mA. The change in the
current response during insertion into the yolk sack was even
more pronounced. This shifted the current further down to
around 0.18 mA and resulted in a pronounced transient response.
This transient response during injection into the yolk sack was
likely caused by the inflow of cellular material into the micro-
injection needle. Once it was expelled, the current response and
the flow were able to reach steady state. The distinct current
amplitude and shape could be used to provide real-time feedback
about the location of the needle for improved targeting and
automation of the device.
Fig. 10 Current–time transient response in un-injected state and during
injection into chorion and yolk sack. The potential applied was 10 V.
This journal is ª The Royal Society of Chemistry 2009
The final step in the characterization of the device was the
measurement of the electroosmotic flow rates. These were char-
acterized using the neutral marker method.28 We characterized
the flow rates by tracking the interface between a clear pH 10
buffer solution and the methylene blue and rhodamine b solu-
tions inside the injection needle. Initially the injection needle was
completely filled with the rhodamine B or methylene blue solu-
tion by engaging the electroosmotic flow. The target channel was
then flushed and care was taken to minimize pressure imbalances
across the needle. Subsequently, a reverse potential was applied
and the rate at which the interface between the solutions moved
inside the needle was measured. The corresponding volumetric
flow rate was calculated using the dimensions of the frustrum
through which the interface moved over a specified time period.
The flowrates that were obtained for the device in an un-injected
state are shown in Fig. 11. They ranged between 9.3 pL/s–
46.5 pL/s for the rhodamine B solution and 2.1 pL/s–14.7 pL/s
for the methylene blue solution, for potentials between 5 V–25 V.
Due to the difficulty in measuring flow rates during injection,
these values were calculated instead. Our method supposes that
the flow rates during injection can be obtained by determining
the change in the potential across the needle caused by the
presence of the embryo and then calculating the electroosmotic
flow rate using this new potential. The Smoluchowski equation
(eqn. 1) was used for this purpose:
uEO ¼ mEO$E (1)
where mEO is the electroosmotic mobility and uEO the electroos-
motic velocity. The velocity was calculated from measured flow
rates, which is a good approximation given the small taper (y/x ¼
0.0094) in the section that was measured. E is the electric field
across the needle, which we calculated using:
I,RN
EN ¼ (2)
lN
where I is the current, lN the total needle length and RN the
resistance across the needle which was obtained from eqn. (3):
RT ¼ RN + RTC + RRC (3)
Fig. 11 EOF rates for rhodamine and methylene blue in un-injected
state.
Lab Chip, 2009, 9, 3202–3211 | 3209
 where RT is the measured total resistance of the system, RTC the
resistance in the target channel and RRC the resistance in the
reagent channel. By solving eqns. (2) and (3) and substituting, we
were able to solve eqn. (1). This provided us with the electroos-
motic mobility of the flow inside the injection needle. The mean
electroosmotic mobility for the rhodamine B flow was found to
be 5.49  103 cm2/Vs. This electroosmotic mobility was then
used to calculate the electroosmotic velocity and flow rate during
injection into an embryo. This was done by first determining the
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resistance across the needle during injection, denoted as RN,Inj.
The resistance was calculated using eqn. (4):
RT,Inj ¼ RN,Inj + RTC + RRC + REmbryo (4)
where RT,Inj is the measured total resistance of the system during
injection, RN,Inj is the potential across the needle during injection
and REmbryo is the added resistance due to the presence of the
embryo, which was obtained from substracting RT from RT,Inj.
The electric field across the needle was then calculated using the
resistance across the needle during injections and the measured
current. This provided us with the effective electric field that the
solution inside the needle experiences. The new electric field in
combination with the electroosmotic mobility of the flow were
then used in eqn. (1) to obtain the flow rate during injection into
the embryo. The calculated flow rates are shown in Fig. 12 and
ranged from 5.1 pL/s–30pL/s which is a 40% reduction compared
to the flow in the uninjected state. This method of calculating the
reagent delivered during injections may be verified using fluores-
cence based volume measurements. It has the potential to enable
highly accurate individualized dosing with real-time electrical
control which compensates for differences between embryos. This
will facilitate highly standardized injections that result in identical
volumes of reagent being delivered into embryos.
Viability of electroosmotic injection
To test the performance of the device, a preliminary viability
study was conducted using Zebrafish embryos in an E3 embryo
Fig. 12 EOF rate in un-injected and injected state (calculated) for
rhodamine. Top axis shows potential drop across embryo during
injection.
3210 | Lab Chip, 2009, 9, 3202–3211
Fig. 13 Zebrafish embryo showing normal development 48 hours after
EOF injection of ultrapure water at 10 V for 10 s.
buffer solution (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2,
0.33 mM MgSO4). The embryos were injected into the yolk sack
with an ultrapure water solution. This combination of buffer and
carrier solution is widely used for Zebrafish injections and thus
provided a realistic working environment. The embryos were
separated to provide a control and samples for injection. A total
of 20 embryos were injected by the application of 10 V for 10 s.
After 48 hours it was found that 75% of the control embryos and
80% of the injected embryos were viable and developed normally
at 48 hours. An image of an injected Zebrafish embryo at
48 hours is shown in Fig. 13. These preliminary results are
promising as they show that EOF may be a viable injection
method.
Another possible application of our device would be in in-vitro
fertilization. However, it will first have to be established whether
the electric field that is required for the electroosmotic reagent
transport has any detrimental effect on the egg and sperm to be
injected. Since electroporation has successfully been used to
transfect mammalian oocytes30 and sperms,31 it is conceivable
that electroosmotic injection will be a viable method as the
electric fields generated are lower than those used in electro-
poration. Alternatively, since scalability and throughput are less
of an issue for in-vitro fertilization, the system could be modified
to employ pressure driven flow for this application. The operator
can still take advantage of the novel compliant injection mech-
anism which provides greater control for injections and reduces
the variability associated with conventional microinjection.
Conclusions
In conclusion, we have successfully fabricated a scalable micro-
injection system and tested it on Zebrafish (Danio rerio)
embryos. The system can be automated to allow for large-scale
clinical applications. The injection mechanism presented greatly
simplifies existing methods as only a single DOF is required for
operation. The electroosmotic reagent delivery mechanism
obviates the restrictions associated with pressure microinjection.
This journal is ª The Royal Society of Chemistry 2009
 We have shown flow control in the pL/s range. However, even
lower rates may be obtained by using smaller injection needles.
Furthermore, we propose a method that will enable highly
accurate and individualized flow control during injections. We
believe that electroosmotic delivery may also result in more
favorable and accurate expressions as it is less invasive than
pressure driven delivery. A preliminary viability study using our
device has resulted in viable embryos. Therefore we believe that
EOF may be a promising new method for reagent transport. We
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are presently working on optimizing the operating parameters to
enable more rapid reagent transport and increased throughput.
Additional viability studies are also currently in progress.
Acknowledgements
We thank Dr. C. Y. Ching and Dr. S Shankar for valuable
discussions. This work was supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC) and the
Ontario Centre of Excellence (OCE).
Notes and references
1 H. Niemann and W. A. Kues, Reproduction Fertility and Development,
2007, 19, 762–770.
2 T. Umezawa, M. Fujita, Y. Fujita, K. Yamaguchi-Shinozaki and
K. Shinozaki, Current Opinion in Biotechnology, 2006, 17, 113–122.
3 K. Takahashi, K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka,
K. Tomoda and S. Yamanaka, Cell, 2007.
4 D. Luo and W. M. Saltzman, Nat. Biotechnol., 2000, 18, 33–37.
5 A. L. Parker, C. Newman, S. Briggs, L. Seymour and P. J. Sheridan,
Expert Reviews in Molecular Medicine, 2004, 5, 1–15.
6 S. Mehier-Humbert and R. H. Guy, Adv. Drug Delivery Rev., 2005,
57, 733–753.
7 Y. Zhang and L.-C. Yu, BioEssays, 2008, 30, 606–610.
8 G. Minaschek, J. Bereiter-Hahn and G. Bertholdt, Exp. Cell Res.,
1989, 183, 434–442.
9 J. R. Walton, J. D. Murray, J. T. Marshall and C. D. Nancarrow,
Biol. Reprod., 1987, 37, 957–967.
10 R. D. Purves, Journal of Neuroscience Methods, 1979, 1, 165–178.
11 R. Purves, Trends in Neurosciences, 1980, 3, 245–247.
This journal is ª The Royal Society of Chemistry 2009
12 S. Zappe, M. Fish, M. P. Scott and O. Solgaard, Lab Chip, 2006, 6,
1012–1019.
13 W. Wang, X. Liu, D. Gelinas, B. Ciruna and Y. Sun, PLoS ONE,
2007, 2.
14 Y. Fukui, E. S. Lee and N. Araki, American Society of Animal
Science, 1996, 74, 2752–2758.
15 P. Mitchell, Nat. Biotechnol., 2001, 19, 717–721.
16 C. H. Hsu, C. Chen and A. Folch, Lab Chip, 2004, 4, 420–424.
17 A. Adamo and K. F. Jensen, Lab Chip, 2008, 8, 1258–1261.
18 S. Lee, W. Jeong and D. J. Beebe, Lab Chip, 2003, 3, 164–167.
19 A. Noori and P. R. Selvaganapathy, in Proceedings of the 12th
International Conference on Miniaturized Systems for Chemistry and
Life Sciences (MicroTAS 2008), San Diego, CA, 2008, pp. 1971–
1973.
20 A. Noori, P. R. Selvaganapathy, in Proceedings of the 5th
International Conference on Microtechnologies in Medicine and
Biology (MMB 2009), Quebec City, Quebec, Canada, 2009,
pp. 142–143.
21 M. M. H. Storms, C. va de Schoot, M. Prins, R. Kormelink,
J. W. M. va Lent and R. W. Goldbach, Plant J., 1998, 13, 131–140.
22 V. Tandon, S. K. Bhagavatula, W. C. Nelson and B. J. Kirby,
Electrophoresis, 2008, 29.
23 M. J. Lukkari, M. I. Karjalainen, R. Sarkanen, M. L. Linne,
T. O. Jalonen and P. J. Kallio, in 26th Annual International
Conference of the Engineering in Medicine and Biology Society,
2004, pp. 2557–2560.
24 J. C. McDonald and G. M. Whitesides, Acc. Chem. Res., 2002, 35,
491–500.
25 A. Noori and P. R. Selvaganapathy, Bonding & Sealing of
components in PDMS by deposition of micro-drops of PDMS
using micropipette, Chips & Tips (Lab on a Chip), 9th September
2008, http://www.rsc.org/Publishing/Journals/lc/Chips_and_Tips/
bonding_and_sealing.asp.
26 A. Ny, M. Autiero and P. Carmeliet, Exp. Cell Res., 2006, 312,
684–693.
27 J. M. Spitsbergen and M. L. Kent, Toxicologic Pathology, 2003, 31,
62–87.
28 K. F. Schrum, J. M. Lancaster, S. E. Johnston and S. D. Gilman,
Anal. Chem., 2000, 72, 4317–4321.
29 K. S. Huang, Y. C. Lin, K. C. Su and H. Y. Chen, Biomedical
Microdevices, 2007, 9, 761–768.
30 J. B. Grabarek, B. Plusa, D. M. Glover and M. Zernicka-Goetz,
Genesis, 2002, 32, 269–276.
31 M. B. Gagne, F. Pothier and M. A. Sirard, Molecular Reproduction
and Development, 1991, 29.
Lab Chip, 2009, 9, 3202–3211 | 3211