|
By Meghana Honnatti and Gareth Hughes, Zyvex Instruments
and Karthik Colinjivadi and J.B. Lee, The University of Texas
at Dallas
Download a printable copy here.
Introduction
Individual cell manipulation has been gaining prominence
in a wide range of applications including stem cell sorting,
gene and molecular delivery, cellular diagnostics, and single
cell-based assays. Direct, physical cell manipulation offers
much more precise selection and understanding of cell properties
than data-averaging over a population of cells. Manipulation
of cells is a challenging task, as it requires not only a
precise, controllable manipulator set-up but also a suitable
end-effector which can be actuated to perform desired tasks
without damaging the cells. Major considerations for performing
manipulations (like gripping and moving of isolated cells)
are biocompatibility of the end effector, the precision of
positioning the end-effector, and gentler handling force.
The Zyvex L200 Nanomanipulation System is a highly versatile
manipulation platform comprised of independently-controllable
nanopositioners which provide sub-micron resolution for precise
and accurate motion control. Combined with suitable end-effectors,
one can use the L200 to manipulate biological
samples — ranging from biopolymers to cells. Polymer
microgrippers made of SU-8, an epoxy-based negative photoresist,
have been fabricated and mounted as end effectors onto the
L200 in order to perform manipulation of cells in suspension.
This application note presents a brief description of the
polymer microgripper, and a demonstration of the manipulation
of suspended cells in aqueous media using these microgrippers.
Cell Manipulation — Overview
Directed, controllable manipulation of cells in an aqueous
medium is attracting interest for biomedical applications.
Various manipulation techniques have been developed to achieve
controlled manipulation of cells. These techniques can be
broadly classified into contact and non-contact techniques.
Non-contact techniques predominantly employ optical principles;
examples include opto-electronic tweezers and laser tweezers.
A major disadvantage of these tweezers is the potential damage
they can cause to biological systems, resulting in a decrease
in the amount of active lifetime of a system — limiting
the time that can be spent studying a system. Other non-contact
techniques utilize electric and magnetic fields. These methods,
though capable of trapping single and multiple cells, are
typically cell-specific and require complex electrode or magnetic
arrangements.
The most generally-used contact technique employs vacuum
technology for holding cells. This technique, capable of holding
cells for micro-injection, doesn’t give sufficient control
for cell sorting and isolation. Another contact technique
for manipulating biological specimens involves the use of
micro-fabricated devices such as microgrippers and microprobes.
Technologies used in micro-electromechanical systems (MEMS)
enable the fabrication of microscale devices which provide
a wide range of options in terms of design, performance, and
material compatibility. Direct motion control of microgrippers
enables controllable cell manipulation and multi-functional
capabilities, including cell sorting, cell isolation, and
cell positioning. Recent developments in fabrication technologies
for polymeric micro-devices have been a significant factor
in the growth of research activities in microscale, biocompatible
tools. Microgrippers can be easily and securely mounted onto
the L200 as robust end-effectors for performing biological
manipulation.
Intracellular pH Sensing
Biological manipulation tools require end-effectors which
are biocompatible, operate at physiological temperature, and
have gentle handling forces. In addition, such tools should
also be capable of being integrated with manipulation systems
to achieve precise, controllable motion. Microgrippers made
of metal or silicon, and those actuated with electrostatic
or piezoelectric mechanisms, do not meet one or more of the
above mentioned criteria.
Electrothermally-actuated polymer microgrippers have been
developed specifically to serve as end-effectors for manipulating
biological specimens.
SU-8, an epoxy-based negative photoresist has been used as
the structural layer, while nickel is used as the heating
layer for the electrothermal actuators. The properties of
SU-8 (such as structural rigidity, high coefficient of thermal
expansion [~52 x 10-6/oC] and ability to make high aspect
ratio structures) have been utilized to achieve grippers with
low power consumption and, consequently, low operating temperature.
The fabricated microgripper is shown in Figure [1a]. The
microgripper consists of one movable arm and one stationary
arm. Long, bent beams, connected in series, act as the electrothermal
actuator. As compared to a normal chevron-type beam, this
design has higher resistance (and consequently lower current
flow), resulting in a lower operating temperature. The electrothermal
actuators (made of nickel) are resistively heated by passing
current. Heat is then transferred onto the bulk SU-8 layer
which expands, producing the required lateral displacement
at the gripper tips. In order to avoid undesired out-of-plane
displacement, the ratio of SU-8-to-nickel thickness is kept
high
(50 ••mm: 2 mm).
The grippers are photolithographically patterned resulting
in highly-repeatable manufacturing. Once patterned, the sacrificial
layer between the SU-8 and the substrate is removed, leaving
behind the grippers which are suspended via tethers. Finally,
the grippers are completely released from substrate (by breaking
the tethers) and are packaged onto a heat dissipating sub-mount
[1b].
Figure 1a (top): Zyvex NanoEffector®
microgripper sub-mount.
Figure 1b (bottom): Polymer microgripper
suspended to pads using tethers, prior to mounting on end-effector.
L200 Setup for Biological Manipulation
Figure [2] shows the set-up for biological manipulation using
the L200 installed on an inverted microscope. The L200 fits
onto the microscope stage using a mounting ring which allows
for arbitrary placement for each of the independently-controllable
positioners. The joystick control of the L200 enables precise
X, Y, and Z manipulation of the end-effector. The mounted
microgripper is loaded as an end-effector onto the L200 system
using a dedicated
adaptor as shown in Figure [3].
Figure 2: Compact L200 control cabinet, 27"
H x 21" W x 26" D (left). L200 installed on a Nikon
TE2000 inverted confocal microscope (right).
Figure 3: Polymer gripper mounted as end-effector
for biological manipulation. Insert shows the ceramic gripper
mount with electrical pads.
Manipulation of Cells in Aqueous Medium
In order to demonstrate single-cell manipulation, normal
rat kidney (NRK) cells, suspended in phosphate buffered saline
(PBS) solution, are dispensed into a petri dish and placed
on the microscope stage. The microscope objective is focused
onto the desired cell to be moved. The nanopositioner mounted
with the polymer microgripper is controlled using the joystick
so that the gripper is brought directly above the desired
cell [4a]. Due to the working distance of the objective lens,
the microgripper is out of the focal plane.
The gripper is normally in the closed position, with the
gap between the arms designed to be similar to the desired
cell diameter. Once a DC voltage is applied, the gripper opens
and remains stable in that state until the voltage is turned
off. The actuated gripper is slowly brought down using the
joystick control so that the gripper tips enclose the cell
[4b]. The DC voltage is then cut off and the gripper closes
and holds the cell [4c]. An advantage of this method is reduced
agitation in the solution during the gripper actuation and
de-actuation. The gripper holding the cell is then slowly
moved to the desired location in the solution, actuated again,
and the cell is slowly released [4d-f].
Figure 4 Sequence of single cell manipulation.
Conclusion
Manipulation of individual cells is desired for single-cell
sorting and cell-based diagnostics. In order to effectively
manipulate cells while maintaining cell viability, precision
manipulators and biocompatible end effectors are necessary.
The L200 Nanomanipulation System mounted on an optical microscope,
and fitted with a polymer microgripper, can perform in vitro
cellular manipulation with sub-micron precision and repeatability
enabling a range of applications where gentle handling and
directed manipulation of living cells are critical.
References
1. Lee, H., et al. Electric and Magnetic Manipulation of
Biological Systems. AIP Conference Proceedings, 2005. 772(1):
p. 1583-1584.
2. Yi, C., et al. Microfluidics technology for manipulation
and analysis of biological cells. Analytica Chimica Acta,
2006. 560(1/2): p. 1-23.
3. Alexandra, B.F., et al. Electronic sorting and recovery
of single live cells from microlitre sized samples. Lab on
a Chip - Miniaturisation for Chemistry & Biology, 2005.
6(1): p. 121-126.
4. Han, S.W., et al. A molecular delivery system by using
AFM and nanoneedle. Biosensors & Bioelectronics, 2005.
20(10): p. 2120-2125.
5. Matsuoka, H., et al. High throughput easy microinjection
with a single-cell manipulation supporting robot. Journal
of Biotechnology, 2005. 116(2):
p. 185-194.
6. Ferrari, E., et al. Biological samples micro-manipulation
by means of optical tweezers. Microelectronic Engineering,
2005. 78-79: p. 575-581.
back to top
| ©
Copyright 2008, Zyvex Instruments. All Rights Reserved. |
|
