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Electrochemical cell lysis device for DNA extraction†

Hun Joo Lee,ab Joon-Ho Kim,*a Hee Kyun Lim,ac Eun Chol Cho,a Nam Huh,a Christopher Ko,a Jae Chan Park,a
Jeong-Woo Choibd and Soo Suk Lee*a
Received 13th August 2009, Accepted 13th November 2009
First published as an Advance Article on the web 17th December 2009
DOI: 10.1039/b916606h

We present a novel electrochemical cell lysis device to prepare DNA samples for lab-on-a-chip (LOC)
applications. It utilizes the electrolysis of saline solution to generate hydroxide ions (OH) at the
cathode as alkaline lytic agents. Cathode and anode chambers are separated by a negatively-charged
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H

ion exchangeable polymer diaphragm to maintain the high pH level for efficient cell lysis in the cathode
chamber, to prevent inflow of PCR-amplification inhibitors from the anode chamber, and to minimize
binding of DNA molecules. Electric current flow and pH maintenance, which depended on the device
design, were two important parameters of the device performance. After optimizing the design and
visually confirming cell lysis of Chinese hamster ovary (CHO) cells in a very short amount of time, we
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directly electrolyzed four bacterial cell types suspended in saline solution. Real-time PCR (qPCR)
analysis showed that our device could lyse both gram-positive and gram-negative bacterial cells with
higher efficiency than other common methods and could detect DNA on the microlitre scale. Our data
demonstrate several advantages of the proposed device: absence of cell lysis chemicals and heating; no
adverse effects on PCR amplification; low DNA loss; low voltage and power consumption; and rapid
processing. The device could potentially be applied as an on-chip DNA extraction component.

Introduction because it would require time-consuming reagent addition steps

Recent advances in miniaturization of various components for
chemical and biological assays have enabled their integration
into lab-on-a-chip (LOC) systems.1–4 One of the major bottle-
necks in traditional bioanalytical methods involves sample
preparation for subsequent detection and analysis, rather than
detection itself. Therefore, development of microfluidic compo-
nents for sample preparation, which is required for complete
automation of diagnostics, has been receiving much attention.5–9
Biological sample preparation often requires access to intracel-
lular biomolecules. Cellular membranes can be disrupted by
various external stresses, such as physical, chemical, mechanical,
electrical and enzymatic methods. The most common technique
for cell lysis is the chemical method since the protocols are well-
established.10 The process was originally described by Birnboim
et al. A suspension of host cells mixed with alkaline (NaOH)
solution of sodium dodecyl suplhate (SDS) solubilized the cell
membrane and caused the release of cellular contents.11,12 The
disadvantage is that it is difficult to apply this technology to LOC
a
Bio & Health Lab, Samsung Advanced Institute of Technology, Samsung
Electronics Co., Ltd., San #14-1, Nongseo-dong, Giheung-gu, Yongin-si,
Gyeonggi-do, Republic of Korea. E-mail: soosuk@samsung.com; mythos.
kim@samsung.com; Fax: +82-31-6816; Tel: +82-31-280-6947; +82-31-
280-6939
b
Interdisciplinary Program of Integrated Biotechnology, Sogang
University, 1 Shinsu-dong, Mapo-gu, Seoul, Republic of Korea
c
Department of Bio and Brain Engineering, Korea Advanced Institute of
Science and Technology, 335 Gwahak-ro, Yuseong-gu, Daejeon, Republic
of Korea
d
Department of Chemical and Biomolecular Engineering, Sogang
University, 1 Shinsu-dong, Mapo-gu, Seoul, Republic of Korea
† Electronic supplementary information (ESI) available: Additional
Table S1 and Fig. S1–S6 as noted in the text. See DOI: 10.1039/b916606h
and complex microfluidic components, including injection
channels and mixers to homogenize the samples.13–19 In addition,
detergents can interfere with downstream assays.20 Numerous
irreversible electroporation methods that can lead to efficient cell
lysis have also been reported.10,21–24 One of the advantages of
these processes is that cell lysis can be very rapid and can easily be
coupled with electrophoresis.21,22 However, these techniques
typically require high field strengths for lysis and can lead to joule
heating of the working fluid.
A new cell lysis method based on an electrochemical process
has been presented by Di Carlo et al.25,26 Electrolysis of phos-
phate buffered saline (PBS) solution generated hydrogen ions at
the anode and hydroxide ions at the cathode, which successfully
disrupted the cell membrane. By cleaving the fatty acid–glycerol
ester bonds in phospholipid molecules, excess hydroxide ions led
to the creation of lysophospholipids and induced membrane
permeabilization.27,28 A significant advantage of this lysis tech-
nique, although not appropriate for retrieving RNA as an
alkaline environment leads to hydrolysis along the length of the
RNA backbone,29 is that local generation of hydroxide ions in
the cell-suspending buffer does not require fluid ports or valves
for reagent delivery and does not dilute sample concentration.
However, electrolyzed solutions produced at the cathode and the
anode must be kept away from each other for numerous reasons.
First, without separation, H+ is spontaneously quenched by
recombination with OH. For efficient cell lysis, high concen-
tration of hydroxide ions should be maintained, as membranes of
certain gram-positive bacterial cells, such as Staphylococcus
epidermidis, are difficult to disrupt.30 In addition, by-products of
electrolysis from the anode, such as hypochlorous acid, have
been suspected as potential inhibitors of DNA synthesis and
PCR amplification.31 Adsorption of DNA extracted from cells to

626 | Lab Chip, 2010, 10, 626–633 This journal is ª The Royal Society of Chemistry 2010

the anode32 can also decrease DNA available for PCR amplifi-
cation.
To solve these problems, we have developed a novel electro-
chemical cell lysis device for DNA extraction with cathode and
anode chambers separated by a negatively-charged ion exchange
polymer diaphragm. After confirming the lytic capability of the
electrolyzed solution generated at the cathode chamber and
improvement of cell lysis and PCR efficiencies in the presence of
the negatively-charged polymer, we optimized the device design
and evaluated lysis efficiency of various bacterial cells suspended
in saline solution in comparison with conventional methods. Our
sample preparation micro-device is a practical approach to be
integrated into LOC for various applications.
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H
Experimental
Reagents
All reagents were received and used without further purification.
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Sodium 4-vinylbenzenesulfonate (SVS), acrylamide, N,N0 -meth-
ylenebisacrylamide (BIS), ammonium persulfate (APS), and 100
mM sodium chloride solution were purchased from Sigma-
Aldrich (St. Louis, MI). N,N,N0 ,N0 -Tetramethylethylenediamine
(TEMED) and PCR grade water were obtained from Promega
(Madison, WI). Brain heart infusion (BHI) broth and nutrient
agar (NA) broth were acquired from Becton, Dickinson and
Company (Franklin Lakes, NJ). CD CHO medium was
purchased from Invitrogen (Carlsbad, CA). LightCycler Fast-
Start DNA Master SYBR Green I, uracil–DNA glycosylase
(UNG) and MgCl2 were obtained from Roche Diagnostics
(Indianapolis, IN).
Cell culture
Bacterial strains used in this study, Escherichia coli (American
Type Culture Collection No. 45020, E. coli), Pseudomonas putida
(American Type Culture Collection No. 12633, P. putida),
Staphylococcus epidermidis Evans (American Type Culture
Collection No. 12228, S. epidermidis), and Streptococcus mutans
Clarke (American Type Culture Collection No. 35668,
S. mutans), were obtained from Korean Collection for Type
Cultures (KCTC; Daejeon, Korea). E. coli and S. mutans were
grown at 37  C with vigorous aeration in 3 mL of BHI media to
an exponential phase (OD600 ¼ 0.5–1.0). P. putida and S. epi-
dermidis were cultured with vigorous aeration in 3 mL of NA
media at 27  C and 37  C, respectively, to an exponential phase
(OD600 ¼ 0.5–1.0). Chinese hamster ovary (CHO) cells, which
were a gift from Professor Gyun Min Lee at Korea Advanced
Institute of Science and Technology (KAIST), were maintained
in CD CHO Medium.
Lysis efficiency of electrolyzed solutions
Electrolyzed solutions were prepared using an electrolysis
apparatus. Two chambers with volume capacity of 10 ml were
separated by Nafion membrane (DuPont, Wilmington, DE),
and positive and negative platinum electrodes were installed in
separate chambers as shown in Fig. S1.† After introduction of
100 mM NaCl solution to each chamber, constant (DC) electric
field of 10 V was applied for 5 min at room temperature.
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Electrolyzed cathode solution (ECS) was obtained from the
chamber with the negative electrode, and electrolyzed anode
solution (EAS) was obtained from the chamber with the positive
electrode. Electrolyzed total solution (ETS) was a 1 : 1 mixture of
ECS and EAS.
E. coli cells were washed twice by centrifugation at 6,000 g for
10 min at 4  C (5180R, Eppendorf, Hamburg, Germany) and
resuspended to densities of 108 cells ml1, 107 cells ml1 106 cells
ml1, or 105 cells ml1 in 100 mM NaCl solution. Aliquots of the
cell suspensions were treated with equal volume of ECS, EAS, or
ETS at room temperature for 5 min. For comparison, cells were
lysed through 5 thermal cycles, consisting of 30 s of 95  C and
30 s of 30  C. Cells that were diluted with equal volume of
100 mM NaCl solution served as our control. These samples were
cultured on LB agar plates at 37  C for 16 h, and lysis efficiencies
of the electrolyzed solutions were assessed by visually comparing
growth of the cells.
Real-time PCR (qPCR) was used as another method to
analyze cell lysis. The assay was performed with a primer set
(forward: 50 -AGTGTGGATTCGGCACTCCT-30 and reverse:
50 -GATTCTTCTTCTAGGGGACCTG-30 )33 to amplify a part
of the core region of the HBV genome inserted into the E. coli
genome using LightCycler 2.0 instrument (Roche Diagnostics).
After appropriate treatment, the samples were centrifuged at
10,000 g for 10 min at 4  C to remove non-lysed cells, and 5 mL of
supernatants, or cell suspensions diluted with equal volume of
saline solution for our control, were mixed with 2 mL of Light-
Cycler FastStart DNA SYBR Green I Master Mix, 3.2 mL of
MgCl2, 1.0 mL of forward and reverse primers (Genotech, Dae-
jeon, Republic of Korea), 4.0 mL of UNG, and 4.8 mL of PCR
grade water. FastStart Taq DNA polymerase was used to
prepare the Master Mix. After distributing to LightCycler
capillaries, UNG was activated at 50  C for 10 min to prevent
carryover contamination between PCRs. Then, the reaction
mixture was initially heated at 95  C for 10 min to denature DNA
templates followed by 35 cycles with a denaturation step at 95  C
for 5 s and an annealing–extension step at 62  C for 15 s. The
PCR amplicon was designed to have 120 base pairs.34
For visualization, 1 mL of the amplified products was analyzed
by Agilent 2100 Bioanalyzer instrument (Agilent Technologies,
Santa Clara, CA).35 DNA 500 Labchips (Agilent Technologies)
were used to detect the proportion of the PCR products. Briefly,
microchannels were filled by pipetting in 9 mL of the gel–dye
mixture into the appropriate well and then forced the mixture
into the microchannels by applying pressure to the well via a 1
mL syringe. The ladder well and samples wells were subsequently
loaded with 5 mL of the DNA size marker mixture plus 1 mL of
either the molecular size ladder or sample. After mixing by
vortex, the chip was immediately inserted into the bioanalyzer
and processed according to the instructions of the manufacturer.
The amount of the PCR products was determined by the relative
ratio of the areas of primer–dimer peaks and PCR amplicon
peaks in the chromatogram.
Direct cell lysis with or without a negatively-charged membrane
To directly confirm whether incorporation of a negatively-
charged membrane and compartmentalization of cell lysis in the
cathode chamber led to significantly improved lysis and PCR
Lab Chip, 2010, 10, 626–633 | 627
 View Article Online

efficiencies, a miniaturized version of the electrolysis apparatus

(cathode/anode volume capacity: 10 mL) described in the
previous section was fabricated. After introduction of E. coli cells
(108 cells ml1) suspended in 100 mM NaCl solution, and saline
solution to the cathode and the anode chamber, respectively,
electrolysis was performed at 5 V and room temperature for 40 or
120 s. The process was done ten times to collect 100 mL of the
sample from the cathode chamber, which was centrifuged at
10,000 g for 10 min at 4  C. Next, Nafion membrane was
removed from the device, and the whole procedure was repeated
with E. coli suspension (108 cells ml1) in 100 mM NaCl solution.
To compare the amount of DNA present in supernatants of the
two different samples after 30 cycles of amplification, qPCR
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H

analysis was performed to amplify a part of the core region of the

HBV genome inserted into the E. coli genome, as described
previously. For our control, E. coli suspensions (108 cells ml1)
were directly introduced PCR mixtures and analyzed.

Device design
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The electrochemical cell lysis device was designed based on the

conclusion that installation of a negatively-charged polymer to
localize cell lysis in the cathode chamber leads to significant lysis
and PCR efficiencies. Our micro-device consisted of two cham-
bers with positive and negative electrodes separated by an ion
exchange polymer diaphragm. The cell suspension was to be
introduced to the cathode chamber for cell lysis, and the saline
solution was to be introduced to the anode chamber. The volume
capacity of the cathode chamber was fixed at 10 mL. A poly-
(dimethylsiloxane) (PDMS) cover with vent holes and a sample
inlet and outlet enclosed the cathode and the anode chambers. In
order to facilitate ventilation of gas produced from electrolysis,
the electrodes were facing up towards the vent holes instead of
standing against the sides. Square holes were made on the elec-
trodes to allow electric current flow. Eight devices varying in size
and shape were designed to improve the device performance. The
‘,’ shape and ‘P’ shape possessed closed and open electrodes,
respectively (Fig. 1a). The shortest distance between the positive
and the negative electrodes was either 0.5 mm or 1 mm with the
electrode width of either 0.5 mm or 1 mm.
Synthesis of ion exchangeable polymer
Free-radical polymerization, with APS and TEMED as initiator
and accelerator, respectively, was used to form the negatively-
charged ion exchangeable polymer, similar in function to
a Nafion membrane. Small amounts of APS and TEMED were
added to aqueous SVS (28 wt% in deionized water), and the
solution was stirred in a glass vessel at 70  C for 2 h (solution A).
Acrylamide (45 wt% in deionized water), BIS (0.1 wt% in
deionized water), APS, and TEMED were mixed (solution B),
and solution A and solution B were quickly mixed together in the
diaphragm vessel of the device to form the diaphragm (Fig. S2†).
Device fabrication
Fig. 1 Design of the electrochemical cell lysis devices. (a) Cross-
sectional and top-down views of the micro-devices. The cathode and the
anode chambers were separated by the ion exchangeable polymer dia-
phragm. The electrodes with two different shapes, ‘,’and ‘P’, were
placed on the glass slide. The PDMS cover had eight vent holes, sample
inlet, and sample outlet. (b) Photographs of the optimized micro-device
(Device No. 7, 9  9  5.5 mm). Scale bar: 2 mm.
an adhesion layer. Square holes were made on the wafer by
sandblasting. Mold bodies to shape the diaphragm vessel and
cover were etched by deep reactive-ion etching (DRIE) tech-
nique. A mixture of siloxane oligomer and cross-linker (Syl-
gard 184, Dow Corning, Midland, MI) was poured into the
molds, and PDMS was cured at 70  C for 1 h. PDMS was then
carefully peeled off the mold. The fluid inlet and outlet, and the
vent holes were punched in the cover. The glass slide with the
patterned electrodes and the PDMS cover were placed in
a plasma chamber (PDC-002 & PDC-FMD-2, Harrick Plasma,
Ithaca, NY) and exposed to 300 mTorr of oxygen plasma for
1 min. The surface-treated PDMS cover was aligned and bonded
to the glass slide. After formation of the ion exchangeable
polymer, the process was repeated to bond the PDMS diaphragm
vessel to the opposite side of the glass slide.

Fabrication of the device proceeded as follows. First, electrodes

Device performance analysis
were photolithographically defined onto 500 mm Pyrex wafers.
 and platinum (Pt, 1,000 A)
Titanium (Ti, 100 A)  were deposited Performance of the electrochemical device was evaluated based
followed by photoresist lift-off in acetone. Titanium was used as on electric current flow to quickly increase pH in the cathode

628 | Lab Chip, 2010, 10, 626–633 This journal is ª The Royal Society of Chemistry 2010

chamber and its ability to maintain high concentration of
hydroxide ions. A constant (DC) electric field of 5 V was applied
to a 100 mM NaCl solution in the device at room temperature for
40 s, which is known to be sufficient for cell lysis and subsequent
release of intracellular materials.36 Electric current flow between
the cathode and the anode chambers at the start of electrolysis
was assessed using a digital multimeter (34410A, Agilent Tech-
nologies). An AR15 pH meter (Fisher Scientific, Pittsburg, PA)
was used to measure the pH of the solution. Efficiency of pH
maintenance was defined as inverse of initial and final pH
difference. The initial pH was measured at the start of electrol-
ysis, and the final pH was taken 1 min after. Three repetitions
were performed with each experiment.
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H
Cell lysis efficiency of the micro-device
Device with the best pH maintenance and electric current flow
was selected to evaluate its cell lysis efficiency. First, cell lysis in
the cathode chamber was visually confirmed by using Nikon
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Eclipse TE 300 fluorescence microscope (Nikon Instruments,
Tokyo, Japan) equipped with a digital camera. CHO cells
(108 cells ml1) were suspended in 100 mM NaCl solution and
electrolyzed at 5 V for 40 s at room temperature. To analyze cell
lysis efficiency, different bacterial strains (E. coli, P. putida,
S. epidermidis and S. mutans) suspended in 100 mM NaCl
solution to densities of 108 cells ml1 were electrolyzed using the
same conditions: the lysate was retrieved from the cathode
chamber through the sample inlet and the initial amounts of
DNA extracted were measured by qPCR, as described previ-
ously. For comparison, a conventional boiling lysis technique
(5 thermal cycles, consisting of 30 s of 95  C and 30 s of 30  C)
and lysis protocol using a commercial Qiagen DNA preparation
kit (13323, Qiagen, Hilden, Germany) were performed and
following centrifugation to remove non-lysed cells, supernatants
were introduced to PCR mixtures for analysis. The latter method
was performed according to the manufacturer’s instructions and
included incubation of cell suspensions with Proteinase K and
lysozyme solution at 37  C for 30 min to break down the
bacterial cell wall. Detergents in the developed solution ensured
complete lysis of the bacteria. As our control, E. coli suspensions
(108 cells ml1) were directly introduced to PCR mixtures without
isolation of supernatants. Real-time PCR assay was performed
with a primer set (forward: 50 -CCCAGACTCCTACGCG-
AGGC-30 and reverse: 50 -GTATTACCGCAACTGCTGG-
CAC-30 )37 to amplify a region of 16S rRNA of the bacterial
genome. After loading, the reaction mixture was initially heated
at 95  C for 10 min to denature DNA templates followed by 35
cycles with a denaturation step at 95  C for 5 s, an annealing step
at 60  C for 15 s, and an extension step at 72  C for 20 s.
Results and discussion
Effects of the electrolyzed solutions on cell lysis
First of all, a 100 mm NaCl solution was used for cell suspension
prior to electrolysis to match the saline concentration in common
bacterial and mammalian cell culture media, and to avoid the use
of components such as phosphate ions, that could interfere with
the nucleic acid structure and decrease PCR efficiency.38,39 In the
electrolysis of NaCl solution, water, which has a higher standard
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reduction potential than Na+, is reduced to hydrogen gas and
OH at the cathode. Therefore, ECS is basic. On the other hand,
Cl possesses higher standard oxidation potential than water and
is oxidized to chlorine gas at the anode. Chlorine gas reacts
readily with water to form hypochlorous acid (HOCl) and H+,
responsible for acidity of EAS. Hypochlorous acid is a weak acid
that dissociates slightly into hydrogen and hypochlorite (OCl)
ions. Very little dissociation of HOCl occurs below a pH of 6.5,
while complete dissociation to OCl occurs above a pH of 8.5.40
Because the disinfection efficacy of HOCl is significantly higher
than that of OCl, a lower pH is preferred for disinfection of
bacteria.41
Cathode reaction ðECSÞ : 2H2 O þ 2e / H2 ðgÞ þ 2OH ðaqÞ
2Cl ðaqÞ /Cl2 ðgÞ þ 2e
Anode reaction ðEASÞ : Cl2 ðgÞ þ H2 O/HOClðaqÞ þ Hþ ðaqÞ þ Cl ðaqÞ
HOClðaqÞ/Hþ ðaqÞ þ OCl ðaqÞ
E. coli cells were tested to investigate effects of different elec-
trolyzed solutions on cell lysis. Fig. S3† shows pictures of LB
agar plates after overnight incubation of the cells. Incubation of
the cells treated with ECS, EAS, ETS, and thermal cycles did not
lead to cell growth on the LB agar plates (Fig. S2a–d†). On the
other hand, incubation of untreated cells resulted in formation of
bacterial colonies (Fig. S2e†). These observations suggest that
E. coli cells were killed by ECS, EAS, and ETS. Since our method
was comparable to conventional alkaline lysis, it was expected
that ECS mediated cell death through lysis. On the other hand,
HOCl in EAS most likely caused bacterial destruction through
degradation of a wide variety of biomolecules, such as DNA,
RNA and proteins, rather than cell lysis.42 The combined effects
of ECS and EAS in ETS killed E. coli cells.
Real-time PCR (qPCR) is an easy method to assess cell lysis
efficiency, as it can measure initial amounts of DNA extracted
from cells after electrolyzed solution treatment. In order to test
the accuracy and reproducibility of qPCR, the intra-assay vari-
ation was determined in triplicate within a LightCycler run.
Plotting fluorescence intensity against cycle number yields qPCR
amplification curve. A threshold for detection of fluorescence
above background is determined. The cycle number at which the
fluorescence crosses the threshold line is the threshold cycle value
(CT). PCR can be divided into 3 phases: background phase,
exponential growth phase, and plateau phase. The exponential
growth phase, where the amount of products increases loga-
rithmically with maximum efficiency of 2 and minimum
efficiency of 1, is described by eqn (1). T0 is the initial amount of
target DNA, K is the amount of target
CT ¼ (1/log E) log T0 + (log K/log E) (1)
DNA at CT, and E is the efficiency of amplification.43 A
standard curve, with CT plotted vertically and log T0 plotted
horizaontally, has a slope of (1/log E); the higher the intial
amount of DNA, the lower the CT value since target DNA level
can be achieved with less PCR cycles. Assuming equivalent PCR
efficiencies for the standard curve and the samples, the initial
amounts of DNA can be computed from CT.
We used DCT (CT_control  CT_sample) as a measure of cell lysis
efficiency of the treatment method compared to our control. To
isolate effects of the treatment method on cell lysis, remaining
Lab Chip, 2010, 10, 626–633 | 629

Fig. 2 Real-time PCR curves of E. coli cells (108 cell/ml) that received
different treatment methods. ECS treatment led to lower CT value than
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H
the conventional boiling lysis technique. EAS treatment resulted in higher
CT value than our control, indicating inhibition of PCR amplification.
(C) ECS-treated cells; (B) EAS-treated cells; (O) ETS-treated cells;
(-- --) boiling-treated cells; (—
—) control-cells.
intact cells were separated, and only supernatants containing
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extracted DNA were amplified. On the other hand, since cell
suspensions were directly introduced to PCR mixtures for our
control, amplification could only have occurred with DNA
extracted through the initial heating step and the denaturation
step of PCR. As shown in Fig. 2 and Table 1, there was
a significant difference in the initial amounts of DNA extracted
from the four treatment methods. The DCT values indicated that
ECS treatment led to higher cell lysis than the conventional
boiling lysis. On the other hand, EAS treatment resulted in
a higher CT value (undetermined) than our control, which meant
that it did not effectively lyse cells to release intracellular mate-
rials or inhibited PCR amplification, or both. In addition, the CT
value of the ETS-treated sample was higher than that of boiling-
treated sample and similar to that of our control. Cell lysis effi-
ciency could have been reduced due to quenching of OH from
ECS with H+ from EAS, and by-products of electrolysis in the
anode chamber could have inhibited PCR amplification. Elec-
trophoresis image and Labchip analysis of PCR product
concentrations, displayed in Fig. S4 and Fig. S5, respectively,†
verified these findings. Concentrations of the PCR amplicons
(around 100 base pairs44) from the ECS treated cells were the
highest whereas the PCR products from the EAS treated cells
were absent at all cell densities. In fact, at cell densities of 106 cells
ml1 and 105 cells ml1, only ECS treatment yielded PCR prod-
ucts after 35 cycles of amplification. In designing the electro-
chemical device, it was important to separate the cathode and the
anode chambers to prevent inflow of PCR inhibitors and to
maximize cell lysis efficiency.
Table 1 DCT values of E. coli cells treated with different methodsabcd
Treatment/cell concentration 108 cells ml1 107 cells ml1
ECS 5.47  0.10 7.19  0.05
EAS N/A N/A
ETS 0.50  0.16 N/A
Boiling 2.89  0.25 6.78  0.48
a b 6 1
Three repetitions were performed. At cell densities of 10 cells ml
and 105 cells ml1, CT_sample values could only be obtained with ECS
treated cells. c DCT ¼ CT_control  CT_sample. d N/A: CT_sample value
could not be obtained.
630 | Lab Chip, 2010, 10, 626–633
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Electrochemical cell lysis with a negatively-charged membrane
To confirm that incorporation of a negatively-charged
membrane in an electrolysis apparatus results in higher lysis
efficiency without inhibition of PCR amplification, cells were
directly lysed in the cathode chamber with or without Nafion
membrane. Amplification of extracted DNA from cells that were
lysed in the presence of the membrane led to significantly greater
DCT values compared to electrolysis in the absence of the
membrane (Table 2, Fig. 3). In fact, removal of Nafion
membrane in the apparatus led to similar CT values to our
control, which reflects ETS treatment and control data in the
previous section. Next, with an increase in lysis time, more cells
were lysed to release higher amounts of DNA, and CT values
decreased. However, the extent of the reduction was not as much
for the cells lysed in the absence of the membrane, possibly
because longer lysis time allowed more recombination of OH
from the negative electrode and H+ from the positive electrode,
thus decreasing concentration of the lytic agents. These results
indicated that installing a negatively-charged membrane in an
electrochemical cell lysis device can improve lysis and PCR
efficiencies by maintaining a high concentration of OH and
preventing inflow of PCR-amplification inhibitors, such as HOCl
and OCl, from the anode chamber. In addition, since DNA
molecules are negatively-charged, such membrane can minimize
sample loss from DNA adsorption to the positive electrode.
Optimization of the device design
In designing our micro-device, the cathode and the anode
chambers were separated by a negatively-charged ion
exchangeable polymer diaphrgam similar in function to
a Nafion membrane. The size and the shape of the device were
varied according to Table S1† in order to improve its perfor-
mance, which was measured by electric current flow between the
chambers to increase pH, and its ability to maintain a high
concentration of hydroxide ions. First, efficiency of pH mainte-
nance depended on the shortest distance between the two
chambers since probability of ion diffusion decreased as paths via
the negatively-charged diaphragm became longer. Entrance of
hydrogen ions from the anode chamber and exit of hydroxide
ions from the cathode chamber could reduce the pH of ECS. In
general, the ‘,’ shape resulted in higher pH maintenance effi-
ciency than the ‘P’ shape as the paths increased by as much as
twice the width of the electrode (Fig. S6a†). Although Device no.
2 showed the lowest pH change of 0.026, the pH changes of ECS
from all eight of the devices were below 0.1. These results indi-
cated that the proposed design could effectively maintain the pH
of ECS, and electric current flow was selected as the critical
factor in optimizing the device performance.
Table 2 DCT values of E. coli cells electrochemically lysed in the cathode
chamberab
Type/lysis time 40 s 120 s
Membrane 6.16  0.47 7.56  0.47
No membrane 1.41  0.01 1.57  0.08
a
Three repetitions were performed. b DCT ¼ CT_control  CT_sample.
This journal is ª The Royal Society of Chemistry 2010

Fig. 3 Real-time PCR curves of E. coli cells (108 cell/ml) electrolyzed
with or without Nafion membrane. Cell lysis in the presence of the
membrane led to higher DCT values than lysis without the membrane.
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H
Longer lysis time decreased CT values with more cells lysed to release
higher amounts of DNA. (C) Cells electrolyzed with Nafion membrane
for 120 s. (B) Cells electrolyzed without Nafion membrane for 120 s.
(-) Cells electrolyzed with Nafion membrane for 40 s. (,) Cells
electrolyzed without Nafion membrane for 40 s. (——) Control cells.
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Electric current is directly related to production of hydroxide
ions as described by eqn (2),
R ¼ i/nFV (2)
which is derived from Faraday’s laws of electrolysis. R is the
reaction rate (assuming an equal generation of hydroxide ions
throughout the cathode chamber) and V is the volume of the
cathode chamber, which was 10 mL. The number of electrons (n)
used per OH is 1, and F is Faraday’s constant.25 Therefore, the
Fig. 4 Sequential microscope images of CHO cell lysis at the cathode chamber of the micro-device. Top: time span of 25 s, 400 (scale bar: 50 mm).
Bottom: time span of 7 s, 1,000 (scale bar: 20 mm).
This journal is ª The Royal Society of Chemistry 2010
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higher the current, the faster the generation of hydroxide ions in
the cathode chamber for cell lysis. Electric current between the
cathode and the anode chambers was affected by the type of
electrolyte, dimensions of the electrodes, and the length and area
of electric current paths. In this experiment, the electrolyte
resistance of the devices was identical. A thicker electrode width
would lead to higher electric current flow. The length and the
area of the path were determined by the shortest distance
between the two chambers and the size of the square hole in the
electrodes, respectively. Shorter length and larger area were
preferred to decrease electrical resistance. From Fig. S6b,† device
no. 7 with an electrode width of 0.5 mm, a path length of 0.5 mm,
and a path area of 7.35 mm2 showed the highest electric current
of 2.1 mA. Images of the micro-device (9  9.5  5.5 mm) are
displayed in Fig. 1b.
Lysis of CHO cells using the optimized device
In order to visually confirm cell lysis performed by device no. 7,
CHO cells suspended in 100 mM NaCl solution were introduced
to the cathode chamber and electrolyzed. Cellular membranes
were clearly deformed and eventually disrupted within a few
seconds (Fig. 4). The device was selected to test lysis efficiencies
of various bacterial cells.
Electrochemical lysis of bacterial cells
Lysis effiencies of different bacterial cells were investigated using
the optimized device (SAIT Device) and compared to
Lab Chip, 2010, 10, 626–633 | 631
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Fig. 5 Real-time PCR curves of four bacterial cells that received different treatment methods. Cell lysis using the SAIT Device resulted in the lowest CT
values except for S. epidermidis. (a) E. coli cell suspension. (b) P. putida cell suspension. (c) S. epidermidis cell suspension. (d) S. mutans cell suspension.
(C) ECS treated cells; (B) Qiagen commercial kit treated cells; (-- --) Boiling treated cells; (— —) Control cells.

conventional boiling lysis technique and a protocol using Conclusions

a commercial Qiagen DNA preparation kit. E. coli and P. putida
were used as typical gram-negative cells whereas S. epidermidis
and S. mutans were used as typical gram-positive cells. According
to Fig. 5 and Table 3, cell lysis using the proposed device yielded
significantly higher PCR amplification signals and lower CT
values than other methods. Except for S. epidermidis, the DCT
values of bacterial cells using the electrochemical device were the
highest. On the other hand, the boiling lysis technique could not
disrupt cellular membranes of P. putida, and the Qiagen kit could
not lyse S. mutans. The DCT values of gram-positive cells were
smaller than those of gram-negative cells since they were more
difficult to lyse due to much greater thickness and density of the
cell membrane.24 However, these results indicated that both
gram-negative and gram-positive bacterial cells could be lysed in
the cathode chamber of the proposed device in short amount of
time with significantly higher efficiency than conventional
methods, and the extracted DNA could undergo PCR amplifi-
cation. Additional advantages of our micro-device include
absence of cell lysis chemicals through local generation of
hydroxide ions, low DNA loss, low voltage and power
consumption, and negligible heating. The device could poten-
tially be applied as an on-chip DNA extraction component, and
its integration with downstream LOC components is being
investigated.
Table 3 DCT values of bacterial cells lysed with different methodsabc
Bacterial SAIT
strain Device Boiling Qiagen kit
We have developed a novel micro-device with an ion exchange-
able polymer diaphragm that can extract DNA from cells
through electrolysis. Effects of different electrolyzed solutions on
cell lysis were investigated, and ECS treatment resulted in higher
efficiency than the conventional boiling lysis technique. Signifi-
cant improvement of cell lysis and PCR efficiencies in the pres-
ence of a negatively-charged membrane was confirmed. In
designing our micro-device, cathode and anode chambers were
separated by a negatively-charged diaphragm to maintain a high
pH level for efficient cell lysis in the cathode chamber, to prevent
inflow of PCR-amplification inhibitors from the anode chamber
and to minimize binding of negatively-charged DNA molecules.
The size and shape of the device were varied to improve the
device performance, which was measured by electric current to
increase pH and its ability to maintain high concentration of
hydroxide ions. After visually confirming the lysis of CHO cells
within a few seconds, the optimized device was used to test lysis
efficiencies of different bacterial cells. Real-time PCR analysis
indicated that the electrochemical device could lyse both gram-
positive and gram-negative cells with significant advantages over
conventional methods, which include microlitre sample volume,
higher lysis efficiencies, absence of cell lysis chemicals and heat-
ing, no adverse effects on PCR amplification, low DNA loss, low
voltage and power consumption, and rapid processing. The
device could potentially be applied as an on-chip DNA extrac-
tion component. Research to integrate the micro-device with
downstream components of LOC is ongoing.

Gram-negative E. coli 7.3  0.07 4.9  0.08 4.0  0.04

P. putida 4.8  0.03 N/A 0.6  0.01 References
Gram-positive S. epidermidis 3.9  0.05 2.8  0.01 5.8  0.13
S. mutans 3.5  0.10 2.4  0.05 N/A 1 M. A. Burns, B. N. Johnson, S. N. Brahmasandra, K. Handique,
J. R. Webster, M. Krishnan, T. S. Sammarco, P. M. Man,
a
Three repetitions were performed. b DCT ¼ CT_control  CT_sample. D. Jones, D. Heldsinger, C. H. Mastrangelo and D. T. Burke,
c Science, 1998, 282, 484–487.
N/A: CT_sample value could not be obtained.
2 B. H. Weigl and P. Yager, Science, 1999, 283, 346–347.

632 | Lab Chip, 2010, 10, 626–633 This journal is ª The Royal Society of Chemistry 2010

3 G. M. Whitesides, Nat. Biotechnol., 2003, 21, 1161–1165.
4 M. Mir o and E. H. Hansen, Anal. Chim. Acta, 2007, 600, 46–57.
5 Y. Huang, E. L. Mather, J. L. Bell and M. Madou, Anal. Bioanal.
Chem., 2002, 372, 49–65.
6 J. Lichtenberg, N. F. De Rooij and E. Verpoorte, Talanta, 2002, 56,
233–266.
7 R. H. Liu, J. Yang, R. Lenigk, J. Bonanno and P. Grodzinski, Anal.
Chem., 2003, 75, 4718–4723.
8 A. J. De Mello and N. Beard, Lab Chip, 2003, 3, 11–19.
9 A. Hatch, E. Garcia and P. Yager, Proc. IEEE, 2004, 92, 126–139.
10 S. Lee and Y. Tai, Sens. Actuators, A, 1999, 73, 74–79.
11 H. Birnboim and J. Doly, Nucleic Acids Res., 1979, 7, 1513–1522.
12 F. J. Meacle, R. Lander, P. Ayazi Shamlou and N. J. Titchener-
Hooker, Biotechnol. Bioeng., 2004, 87, 293–302.
13 P. C. H. Li and D. J. Harrison, Anal. Chem., 1997, 69, 1564–1568.
14 E. A. Schilling, A. E. Kamholz and P. Yager, Anal. Chem., 2002, 74,
1798–1804.
Published on 17 December 2009 on http://pubs.rsc.org | doi:10.1039/B916606H
15 G. Ocvirk, H. Salimi-Moosavi, R. J. Szarka, E. A. Arriaga,
P. E. Andersson, R. Smith, N. J. Dovichi and D. J. Harrison, Proc.
IEEE, 2004, 92, 115–125.
16 J. El-Ali, S. Gaudet, A. Gunther, P. K. Sorger and K. F. Jensen, Anal.
Chem., 2005, 77, 3629–3636.
17 C. J. Easley, J. M. Karlinsey, J. M. Bienvenue, L. A. Legendre,
M. G. Roper, S. H. Feldman, M. A. Hughes, E. L. Hewlett,
Downloaded by Duke University on 17 January 2013
T. J. Merkel, J. P. Ferrance and J. P. Lander, Proc. Natl. Acad. Sci.
U. S. A., 2006, 103, 19272–19277.
18 B. Huang, H. K. Wu, D. Bhaya, A. Grossman, S. Granier,
B. K. Kobilka and R. N. Zare, Science, 2007, 315, 81–84.
19 Y. S. Huh, J. H. Choi, T. J. Park, Y. K. Hong, W. H. Hong and
S. Y. Lee, Electrophoresis, 2007, 28, 4748–4757.
20 A. Abolmaaty, M. G. El-Shemy, M. F. Khallaf and R. E. Levin,
J. Microbiol. Methods, 1998, 34, 133–141.
21 F. Han, Y. Wang, C. E. Sims, M. Bachman, R. Chang, G. P. Li and
N. L. Allbritton, Anal. Chem., 2003, 75, 3688–3696.
22 M. A. McClain, C. T. Culbertson, S. C. Jacobson, N. L. Albritton,
C. E. Sims and J. M. Ramsey, Anal. Chem., 2003, 75, 5646–5655.
23 J. Gao, X. F. Yin and Z. L. Fang, Lab Chip, 2004, 4, 47–52.
24 P. R. Gascoyne and J. V. Vykoukal, Proc. IEEE, 2004, 92, 22–42.
25 D. Di Carlo, C. Ionescu-Zanetti, Y. Zhang, P. Hung and L. P. Lee,
Lab Chip, 2005, 5, 171–178.
This journal is ª The Royal Society of Chemistry 2010
View Article Online
26 J. T. Nevill, R. Copper, M. Dueck, D. N. Breslauer and L. P. Lee, Lab
Chip, 2007, 7, 1689–1695.
27 K. Woo and J. Kim, J. Chromatogr., A, 1999, 862, 199–208.
28 J. Arnhold, A. Osipov, H. Spalteholz, O. Panasenko and J. Schiller,
Biophys. J., 2002, 1572, 91–100.
29 R. E. Farrell, in RNA Methodologies: A Laboratory Guide for
Isolation and Characterization, Elsevier Academic Press, London,
3rd edn, 2005, ch. 1, pp. 13–14.
30 B. M. Chassy and A. Giuffrida, Appl. Environ. Microbiol., 1980, 39,
153–158.
31 S. M. McKenna and K. J. A. Davies, Biochem. J., 1988, 254, 685–692.
32 T. Okada, T. Kaneko, R. Hatakeyama and K. Tohji, Chem. Phys.
Lett., 2006, 417, 288–295.
33 Y. K. Cho, J. Kim, Y. Lee, Y. A. Kim, K. Namkoong, H. K. Lim,
K. W. Oh, S. Kim, J. Han, C. Park, Y. E. Pak, C. S. Ki,
J. R. Choi, H. K. Myeong and C. Ko, Biosens. Bioelectron., 2006,
21, 2161–2169.
34 R. W. Chen, H. Piiparinen, M. Sepp€anen, P. Koskela, S. Sarna and
M. Lappalainen, J. Med. Virol., 2001, 65, 250–256.
35 C. Lu, D. Tso, T. Yang, Y. Jong and Y. Wei, Clin. Chim. Acta, 2002,
318, 97–105.
36 Z. Wang, G. Le, Y. Shi and G. Wegrzyn, Process Biochem., 2002, 38,
199–206.
37 H. Hwang, H. K. Kim, S. Y. Jung, K. Namkoong, J. H. Kim, N. Huh,
C. Ko and J. C. Park, Anal. Chem., 2008, 80, 7786–7791.
38 E. V. Pelt-Verkuil, A. Belkum and J. P. Hays, in Principles and
Technical Aspects of PCR Amplification, Springer Science, 2008, ch.
4, pp. 38.
39 S. Dames, L. K. Bromley, M. Herrmann, M. Elgort, M. Erali,
R. Smith and K. V. Voelkerding, J. Mol. Diagn., 2006, 8, 16–21.
40 Handbook of Public Water Systems, John Wiley & Sons, New York,
2nd edn, 2001.
41 T. Oomori, T. Oka, T. Inuta and Y. Arata, Anal. Sci., 2000, 16, 365–369.
42 J. M. Albrich, C. A. McCarthy and J. K. Hurst, Proc. Natl. Acad. Sci.,
1981, 78, 210–214.
43 B. Øster and P. H€ ollsberg, Biol. Proced. Online, 2002, 4, 88–93.
44 The PCR amplicon was designed to have 120 base pairs. Considering
that DNA 500 Labchips used with Agilent 2100 Bioanalyzer have the
10% coefficient of variation (CV), it is reasonable that the bands
around 100 base pairs in Fig.S4† are those of the PCR amplicons.
Lab Chip, 2010, 10, 626–633 | 633