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Ultrasonication on a microfluidic chip to lyse single and multiple Pseudo-nitzschia

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Ultrasonication on a microfluidic chip to lyse single and multiple Pseudo-nitzschia
Biotechnology
Journal
DOI 10.1002/biot.201000224
Biotechnol. J. 2011, 6, 150–155
Technical Report
Ultrasonication on a microfluidic chip to lyse single and multiple
Pseudo-nitzschia for marine biotoxin analysis
Chunsheng Wu1, Peter B. Lillehoj2, Leyla Sabet2, Ping Wang1 and Chih-Ming Ho2
1Biosensor
National Special Laboratory, Key Laboratory for Biomedical Engineering of Ministry of Education, Department of
Biomedical Engineering, Zhejiang University, Hangzhou, China
2 Mechanical and Aerospace Engineering Department, University of California, Los Angeles, CA, USA
We present a microfluidic platform, which provides a simple and efficient means for handling and
processing Pseudo-nitzschia, a neurotoxin-producing marine algae. Currently, analyzing the production of such toxins is complicated by multiple environmental factors and high variability among
individual Pseudo-nitzschia species. To address this issue, we developed a device that can precisely trap single and multiple cells for subsequent lysis to extract relevant intracellular molecules. Our
results show a cell trapping efficiency of up to 96%, which is achieved by hydrodynamic flow focusing. Additionally, complete cell lysis via ultrasonication can be accomplished within a few seconds. This platform can be applied to other algae and non-algae cell types with minimal modification, thus providing a valuable tool for studying biological intracellular mechanisms at the single and multi-cell level.
Received 30 June 2010
Revised 6 December 2010
Accepted 9 December 2010
Supporting information
available online
Keywords: Hydrodynamic cell trapping · Microfluidics · Pseudo-nitzschia · Ultrasonic cell lysis
1 Introduction
In recent years, phytoplankton blooms containing
toxic species of Pseudo-nitzschia have become
more prevalent due to increased pollution of
coastal waters, especially along the western U.S.
coast, posing a serious threat to the health of sea
mammals, sea birds, and humans [1]. Pseudonitzschia is a cosmopolitan genus of pinnate diatoms restricted to marine environments [2] whose
cell structure consists of two long and slender overlapping silica halves [3]. Previous studies have
demonstrated that several Pseudo-nitzschia species are capable of producing domoic acid (DA),
Correspondence: Professor Chih-Ming Ho, Mechanical and Aerospace
Engineering Department, University of California, 420 Westwood Plaza,
Engineering IV, Room 38-137J, Los Angeles, CA 90095-1597, USA
E-mail: [email protected]
Fax: +1-310-206-2302
Abbreviations: ASP, amnesic shellfish poisoning; DA, domoic acid;
GA, glutamic acid; PDMS, poly(dimethylsiloxane)
150
which is one of the most hazardous marine biotoxins known to man [4–7]. Specifically, DA is a neuroexcitatory toxin that acts as a causative agent for
amnesic shellfish poisoning (ASP) in humans
through the ingestion of contaminated shellfish [8].
The chemical structure of DA is similar to the excitatory neurotransmitter glutamic acid (GA), but has
a much stronger receptor affinity, which is up to 100
times than that of GA. When ingested, DA can predominately bind to N-methyl-D-aspartate (NMDA)
receptors in the central nervous system, which will
eventually lead to neuronal swelling and cell death
[9]. Since the first documented case of ASP in 1987
in Prince Edward Island (Canada), which caused at
least three deaths and sickened over a 100 people
[10], Pseudo-nitzschia has been recognized to be of
increasing environmental, economic, and public
health importance. Considerable effort has been
devoted to studying the mechanisms of DA production by Pseudo-nitzschia. Currently, the production
of DA by Pseudo-nitzschia is known to be influenced by several environmental factors, such as the
depletion of silicon [10], a shortage of phosphorus
[11], and the availability of iron [6] in marine wa-
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2011, 6, 150–155
ters. However, understanding DA production is further complicated by high variability between different Pseudo-nitzschia species and individual
cells. Recent research has shown that cells within
the same population exhibit substantial intracellular differences, due to various causes including cellular degradation and stochastic effects in gene expression [12]. Such differences ultimately lead to
fluctuations in the amount of proteins that are produced by a specific gene. Therefore, obtaining a
better understanding of the biological mechanisms
of DA production by Pseudo-nitzschia requires
studies at the single and multi-cell level, which
avoids the loss of information associated with ensemble averaging based on large cell population
studies.
Recent advancements in micro-electro-mechanical systems (MEMS) have allowed for development of devices for handling and processing biological cells at the single-cell level [13, 14]. However, DA extraction in Pseudo-nitzschia at the single-cell level is a difficult task owing to its unique
morphology, rigid crystallized silica cell membrane,
and lack of tools for such processing. In the recent
decade, microfluidic platforms have demonstrated
to be a convenient and promising approach for single-cell studies [15, 16], which have the advantages
of lower reagent consumption, faster analysis
times, and higher temporal resolution compared
with benchtop laboratory methods. Such platforms
provide novel methods for handling and analyzing
single cells from bulk cell suspensions. Current
laboratory methods for cell lysis include the use of
detergents and high temperatures; however, such
methods can influence downstream cell assays as a
result of chemical contamination and molecular
denaturation. Therefore, we propose to use mechanical forces via ultrasonic waves to rupture
Pseudo-nitzschia for DA extraction. Previous work
has demonstrated the versatility of ultrasonicbased methods for cell lysis, which appears to be a
promising approach for automated fluidic biodetection systems [17, 18].
We present a microfluidic-based technique to
trap and lyse single and small populations of Pseudo-nitzschia, enabling for rapid DA extraction. Microcages are positioned at specific locations within
a microchannel and hydrodynamic focusing [19]
allows for cells to be precisely guided into the microcages. This device is coupled with an ultrasonic
lysis method to rapidly rupture the cells. In addition to its portability and single-cell capability, this
technique avoids the common problems of conventional cell lysis methods (chemical reagent contamination, excessive heating, etc.), which can
damage the intracellular components. Ultimately,
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.biotechnology-journal.com
this device has great potential to be incorporated
into an automated biodetection system for rapid infield DA detection.
2
2.1
Materials and methods
Microfluidic chip design and fabrication
The proposed device (Fig. 1a) is designed for accurate single and multi-cell trapping of Pseudonitzschia by adopting the following two strategies.
First, hydrodynamic focusing is utilized to precisely control the cell’s position within the microchannel. The microfluidic chip is composed of three inlets: a center inlet (500 μm in width) is used to load
the cells and two outer inlets (300 μm in width) are
loaded with a buffer solution for flow/cell focusing.
By controlling the flow rates of both the sample and
buffer solutions, the cells can be focused in the center of the channel. The converging fluid flows create a velocity shear, which aligns the cells in the
longitudinal direction, guiding them into the microcages. Second, the microcages (50 μm long and
20 μm wide), which are positioned in the center of
Figure 1. (a) Photograph of the microfluidic chip with three inlets for cell
and buffer solutions. The inset shows an enlarged view of microcages for
cell trapping. Colored dye is flowed into the device for better visulazation
of the channel features. (Scale bar = 1 mm; inset scale bar = 100 μm).
(b) Schematic diagram of the experimental setup.
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Biotechnology
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main microchannel downstream from the inlets,
are designed to accommodate for the unique size
and shape of Pseudo-nitzschia. As shown in Figure
S1 (Supporting Information), Pseudo-nitzschia
cells are long and slender with aspect ratios
(length:width) of at least 10:1. As such, these cells
cannot be caught using current microfluidic traps,
which accommodate for spherical-shaped cells,
and required a new design.
The microfluidic chip was fabricated by assembling a poly(dimethylsiloxane) (PDMS) cast with a
glass substrate. Briefly, SU-8 photoresist (MicroChem, Newton, MA) was spin-coated onto a
cleaned silicon wafer. UV lithography was used to
pattern the SU-8 to form micro-features as an etch
mask, which was baked at 150 °C for 5 min. Next,
deep reactive ion etching (DRIE) was used to etch
into the silicon wafer, forming the PDMS mold. The
depth of the microstructures was approximately
50 μm. PDMS pre-polymer and crosslinker (Sylgard 184, Dow Corning, Midland, MI) were mixed at
a weight ratio of 10:1, degassed, poured on the
wafer, and baked at 80°C for 2 h. After hardening,
the PDMS was peeled off and the inlets and outlets
were punched using blunt-tip needles (Corning,
Lowell, MA). The PDMS cast and a glass slide were
exposed to an air plasma (Harrick, Ithaca, NY) and
then assembled immediately to form an irreversible bond. Colored dye was flowed into the device to test for any leaking.
2.2
Cell culture and reagents
Pseudo-nitzschia and sterile seawater were provided by Dr. Caron’s laboratory at the University of
Southern California. The growth media, 50× Guillard’s (F/2) marine water enrichment solution, was
purchased from Sigma–Aldrich (St. Louis, MO,
USA) and cell culture flasks with vented cap were
purchased from Fisher Scientific (Tustin, CA,
USA). The cell culture media were mixed by diluting the Guillard’s (F/2) marine water enrichment
solution using sterile seawater (also provided by
Dr. Caron’s laboratory) at the ratio of 1:50. Cells
were incubated in a mini-refrigerator at 17–20 °C. A
lamp was installed inside the refrigerator to generate sufficient light for cell growth. The lamp was
controlled by a programmable timer and was set at
12 h light-dark cycles. The cell culture media were
refreshed every 2 wk. The concentration of cells
was adjusted to 105 cells/mL for experiments,
which was measured using a hemacytometer (Gibco, UK). Controlling the cell concentration allowed
for trapping events to occur within a timely manner
as well as minimized cell blockage within the microchannel.
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Biotechnol. J. 2011, 6, 150–155
2.3
Experimental setup
A schematic diagram of the experimental setup is
shown in Fig. 1b. A Branson 2510 ultrasonicator
(Danbury, CT) was utilized for cell lysis. Short
pieces of silicon rubber tubing were glued to the inlets and outlets of the microfluidic chip, which
served as connectors for 1/16” OD Teflon tubing.
Two syringe pumps (Harvard Apparatus, Holliston,
MA) were used for our experiments; one was connected to the sample inlet and the other was connected to the buffer inlets using a plastic T-connector. Experiments were performed under an inverted microscope (Leica, USA) equipped with a Sony
DFW-X710 color CCD camera. Images of cell trapping and lysis were captured using IC Capture image acquisition software (Imaging Source, Charlotte, NC).
3
3.1
Results and discussion
Cell culture
Pseudo-nitzschia is characterized by a unique reproduction cycle where they reproduce both sexually and asexually. Cells primarily undergo asexual
reproduction, which results in a smaller cell size
with each reproduction cycle. When cells are diminished to about 30% of their original size, they
undergo sexual reproduction and regenerate to a
large cell size [20–22]. Pseudo-nitzschia requires
both stable lighting and temperature for proper
growth and health. In our cell culture system, we
used a mini-refrigerator to regulate the temperature at 17–20 °C. At temperatures outside of this
range, the growth speed decreased until the cells
could no longer maintain a sufficient population
size. A timer-controlled lamp was used to mimic
sunlight, which was programmed at 12 h light-dark
cycles. Excess light exposure caused photoinhibition while minimal light exposure inhibited the
cells from receiving enough energy for healthy
growth. Unhealthy and dead cells, which appeared
as translucent silica membranes, were observed after keeping the cell culture chamber dark for more
than 24 h. Additionally, the frequency of replenishing the culture media was observed to be an important parameter. Usually, the culture media becomes
turbid and yellowish-brown in color during later
stages of the growth phase, which indicates that the
media needed to be refreshed. Through optimization of these growth parameters, we were able to
obtain healthy cells, which were characterized by a
long and slender morphology, translucent pigmentation, and the formation of chain colonies (Fig. S1).
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Biotechnol. J. 2011, 6, 150–155
www.biotechnology-journal.com
3.2 Influence of hydrodynamic focusing on cell
trapping
To investigate the influence of hydrodynamic focusing on cell trapping, different degrees of hydrodynamic focusing were generated by controlling the flow rates of the cell and buffer solutions.
With different degrees of hydrodynamic focusing,
the orientation and alignment of the cells in the
microchannel were affected, which directly influenced the cell trapping performance (Figure S2 in
Supporting information). As shown in Fig. S2a,
when there is no hydrodynamic focusing (cell solution flow rate of 5 μL/h), the cells can flow
around the microcages and avoid being trapped.
Figure S2b shows the effects of low hydrodynamic
focusing (cell and buffer solution flow rates of 5
and 10 μL/h, respectively) which can focus the
cells toward the center of the microchannel, but
cannot control their orientation. In this situation,
cells were usually trapped outside of the microcages, leading to the channel becoming clogged.
Figure S2c shows the effects of high hydrodynamic focusing (cell and buffer solution flow rates of 5
and 15 μL/h, respectively) which allowed for proper cell alignment and orientation and for the cells
to be fully trapped inside the microcages. The cell
trapping performance is evaluated by the ratio of
the number of cells trapped inside microcages to
the total number of cells flowed inside the device.
Figure 2 shows statistical results of cell trapping
performance which shows that the trapping efficiency increases with faster buffer flow rates. A
cell trapping efficiency of 96% was achieved at a
buffer flow rate of 15 μL/h and a cell solution flow
rate of 5 μL/h. However, above this threshold velocity, the volume of buffer solution greatly exceeds that of the cell solution, preventing cells
from flowing through the main channel. When the
buffer flow rate is further increased to 20 μL/h, the
cell solution is fully pinched off from the main
channel, inhibiting cells from entering the channel. Therefore, a buffer flow rate of 15 μL/h is optimal for high trapping efficiency and high
throughput.
3.3 Hydrodynamic trapping of single-cell and
multiple cells
In our device, three microcages are sequentially
positioned within the microchannel for cell trapping. Such a design improves the efficiency of cell
trapping: in most cases, with high hydrodynamic
focusing, cells can be trapped by the first microcage. However, if a cell bypasses the first microcage, it can be trapped by the second or third mi-
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Figure 2. Statistical results of cell trapping efficiency at various buffer flow
rates with the sample flow rate kept constant at 5 μL/h. The mean and SD
of four experiments are shown.
crocage. Using our device, both single and multiple
cells can be trapped inside the microcages when a
high degree of hydrodynamic focusing is applied.
The number of trapped cells can be controlled by
the duration of flow; longer durations of flow result
in a greater number of trapped cells. Additionally,
the trapped cells are contained within the microcages once the flow is stopped. Typically, each
microcage can hold up to six cells without having a
negative impact on the flow. When more than six
cells are trapped inside a single microcage, the flow
inside the channel becomes disrupted, leading to a
loss of hydrodynamic focusing. Consequently, the
cells can no longer be focused toward the microcages, diminishing the trapping efficiency.
3.4
Ultrasonic lysis of cells
Once the cells are trapped inside the microcages,
the sample flow is stopped while the buffer solutions continue to flow to flush out any residual cells
from the channel and prevent the trapped cells
from being expelled out of the microcages. The device is then placed in an ultrasonication bath, allowing for ultrasonic waves to propagate within the
microchannel to rapidly lyse the trapped Pseudonitzschia cells. Fig. 3a, b and c show the process of
a single cell being lysed while Fig. 3d, e and f show
multiple cells being lysed. To verify the efficacy of
ultrasonication, we present sequential images at
three instances during the cell lysis process: prior
to ultrasonication (Fig. 3a, d); midway through ultrasonication (Fig. 3b, e) and post-ultrasonication
(Fig. 3c, f). Particularly, images taken midway
through ultrasonication (Fig. 3b, e) show cells being
partially lysed, where the silica shell begins to disintegrate. With further ultrasonication, the silica
membranes of Pseudo-nitzschia are fully disrupted
into small pieces, allowing for the intracellular
components to be released within the channel. The
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Figure 3. Typical results of cell lysis by ultrasonication; (a), (b), and (c)
show a single cell being lysed; (d), (e), and (f) show a cluster of cells being lysed. The time interval between each inset is 3 s. (Arrows denote the
cell’s position; scale bar = 100 μm).
resulting solution can be easily collected for biomolecular detection, where the desired single or
multiple cells are analyzed. As far as the three sequentially positioned microcages are concerned,
the effect of ultrasonication is identical. Based on
our results, cells could be completely lysed in less
than 10 s, which is up to 10 times faster compared
with conventional methods [8]. In addition, this
method does not require the use of any harsh detergents or lysing reagents, which can influence
subsequent detection measurements.
4
Concluding remarks
We present a microfluidic platform for precise cell
trapping combined with rapid ultrasonic lysis of
Pseudo-nitzschia. This technique provides a simple
and efficient tool for handling and processing
Pseudo-nitzschia at the single and multi-cell level,
which can improve our understanding of the biological mechanisms responsible for DA production
when combined with a portable biodetection system. After lysing the cells, the intracellular molecules can be extracted and transported directly to
sensors via microchannels for the downstream detection and analysis. This will greatly facilitate the
realization of an automated and miniaturized biodetection system for in-field DA monitoring. Additionally, the proposed platform can be applied to
other algae and non-algae cell types with minimal
modification, thus providing a valuable tool for
studying biological intracellular mechanisms at the
single and multi-cell level.
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This work was supported by the Center for Embedded Networked Sensing (CENS) through the National Science Foundation of U.S.A. (Grant No. CCR0120778), the China National Funds for Distinguished Young Scientists (Grant No. 60725102), the
National Natural Science Foundation of China
(Grant No. 31000448), the China Postdoctoral Science Foundation (Grant No. 20100471737), and the
Joint Ph.D. Training Program funded by the China
Scholarship Council.We thank Beth Stauffer, Erica L.
Seubert, and Dr. Dave Caron from the University of
Southern California for providing Pseudo-nitzschia
cells and technical support for cell culturing. We also
thank Ieong Wong for his helpful discussion in reviewing the manuscript.
The authors have declared no conflict of interest.
5
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