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Microchim Acta (2010) 170:283288

DOI 10.1007/s00604-010-0370-9

ORIGINAL PAPER

Real-time detection of food-borne bacterial adenosine


triphosphate (ATP) using dielectrophoretic force
and a bioluminescence sensor
Hui-Sung Moon & Hee Taek Im & Ahmi Choi &
Hyo-Il Jung

Received: 30 November 2009 / Accepted: 19 April 2010 / Published online: 19 May 2010
# Springer-Verlag 2010

Abstract Real-time detection and quantification of foodborne bacteria draws increasing interest for evaluation of
food quality and safety. Since living cells invariably contain
adenosine triphosphate (ATP), the detection of bacterial
ATP presents a fascinating method to determine its
presence in food. Care must be taken however, to remove
food-derived extracellular ATP, which will interfere with
detection by ATP-luminescence. We developed a microfluidic and dielectrophoretic (DEP) device for intracellular
ATP detection, which captures microorganisms by DEP
force and washes extracellular ATP away. The yield of
capture by DEP force at a 10 Lmin1 flow rate used in
experiments was 87.7%. At constant ATP level the
electrical sensor responded in proportion to the bacterial
concentration. With a constant bacterial concentration and
varying ATP, the signal did not change. These results show
that the device can remove the extracellular ATP contribution from food to be sampled.
Keywords Real-time detection . ATP(adenosine
triphosphate) . Luminescence . Dielectrophoresis .
Microfluidics

H.-S. Moon : H. T. Im : H.-I. Jung


Laboratory of Biochip Technology,
School of Mechanical Engineering, Yonsei University,
Seoul 120-749, South Korea
A. Choi : H.-I. Jung (*)
National Core Research Center for Nanomedical Technology,
Yonsei University,
Seoul 120-749, South Korea
e-mail: uridle7@yonsei.ac.kr

Introduction
Real-time detection of water-, food-, and airborne microorganisms can potentially forestall outbreaks of food
poisoning and respiratory infections [1]. Microbial contamination of food and kitchen utensils is especially
frequent and hazardous. Conventional detection by colony
counting requires an incubation of several days and
formation of visible colonies. Although the polymerase
chain reaction (PCR) can reliably identify some foodborne pathogens (e.g., Salmonella, E.coli O157, and
Listeria), it requires tedious sample pretreatments, significant processing time, and skilled technical support. An
early warning system for public health awaits more rapid
and effective detection methods. Various immunoassays,
molecular biological tests, and optical and electrical
methods, proposed over several decades [24], have
required too much time, expensive reagents, or complicated equipment.
Adenosine triphosphate (ATP), a universal energy
source, can be quantified in living cells within minutes
using the firefly luciferin-luciferase reaction. This sensitive,
uncomplicated assay for ATP bioluminescence has been
used to detect water- and food-borne microorganisms [58],
and we have used an ATP-bioluminescence transducer to
detect airborne bacteria in real time [9].
In food handling situations, food remnants and organic
debris not only support microbial growth but may also
contribute extracellular ATP [10], which leads to overestimation of bacterial ATP content [5]. To maintain the
accuracy of the bacterial assay, this extraneous ATP is
generally removed by enzymatic digestion [7, 11]. These
methods, however, risk degradation of intracellular ATP,
require impractical conditions of temperature and pH [12,
13] and may not justify the cost.

284

H.-S. Moon et al.

In this paper we present a novel method to remove


extracellular ATP and measure bacterial concentration. We
developed a microelectrode integrated microfluidic device
to capture bacteria and extract ATP only from the bacteria.
The device uses dielectrophoretic force to capture the cells
and an optical sensor to detect their ATP content.
Dielectrophoresis(DEP) refers to the movement of a
particle in an electric field gradient which results from
the difference in polarizability between the particle and the
medium [14]. Particles are attracted to regions of stronger
electric field when their permittivity is higher than that of
the suspension medium. If permittivity of particles is
lower than that of the medium, particles are repelled from
regions of stronger electric field [15, 16]. The DEP force
acting on a spherical particle can be described by the
following equation,
FDEP 2p"m r3 Ref CM rjEj2

where r is the radius of the particle, m is the medium


permittivity, Re[fCM] is the real part of the ClausiusMossotti (CM) factor and E is the electric field intensity.
The CM factor is described as:

 


f CM " p  " m = " p 2 " m

where *p is the complex permittivity of the particle and


*m is the complex permittivity of the medium. The
complex permittivity is then described as:

" "  js=w

where is the relative permittivity (or dielectric constant),


is the electric conductivity, j is (1) and is the
angular frequency of the AC electric field. Particles are
attracted to a strong electric field when the CM factor is
positive, which called positive DEP and to a weak electric
field when the CM factor is negative, which called
negative DEP.
This technique is especially useful in a microfluidic
device, since low voltages will produce significant forces
on cells without contact, modification, or labeling [17].
When the mixture contains both bacterial particles and
extracellular ATP, it is possible to capture bacterial particles
by DEP in certain conditions. We already successfully
isolated bacterial particles from micro-bead or dust mixtures solution [18]. Hence we combined the DEP phenomenon with a microfluidic channel to create a new method
that washes extracellular ATP away. Schematic diagram of
the extracellular ATP removal process is described in
Fig. 1. Our technique can contribute to the development
of a real-time monitoring system for microorganisms in the
food industry.

Experimental
Device fabrication
Our microbial detection device (Fig. 1(ii)) consists of
two parts, a microfluidic channel (top) and a DEP
electrode (bottom). The microfluidic channel is made
from PDMS (polydimethylsulfoxane, Dow Corning,
USA, www.dowcorning.com) polymer and the DEP
electrode part has gold electrodes on a glass wafer.
Microfluidic channels were molded by a soft lithography
process. The organic resin SU-8 2025 (Microchem Corp,
USA, www.microchem.com) was spin-coated to a height
of 50 m, which is equal to the channel depth, on
Si wafers. The wafer was exposed to UV light with a
photo-mask that had a channel pattern. The SU-8 layer
was etched using SU-8 developer and rinsed with isopropyl alcohol. PDMS prepolymer mixture (PDMS:
curing agent = 10:1 w/w, Dow Corning, USA, www.
dowcorning.com) were poured on the SU-8 mold and
degassed using a vacuum chamber. The PDMS was then
cured for 2 h at 80 C in an oven and peeled off. The
PDMS molds were diced by razor blade, and inlet and
outlet holes were made with a punch. The DEP electrodes
were fabricated on a 4-inch glass wafer using a conventional photolithography process. Titanium and gold layers
were deposited on the wafer for electrodes with 200
and 3,000 thickness using sputter. Titanium was used
as an adhesion layer. Photoresist (PR, AZ1512, Germany,
www.microchemicals.com) was spin-coated to 1 m
thickness, then exposed under UV light and developed
to define the electrode patterns. Gold etchant and
buffered oxide etchant (BOE) were used successively to
define electrodes, and acetone was used to remove the
patterned PR. Finally, after O2 plasma treatment for 30 s,
the PDMS microchannel was assembled with bottom
electrodes patterned on glass wafer and stored overnight
in an oven to ensure permanent bonding. Wires were
attached to the electrodes using silver paste.
Measurements of ATP
In the microfluidic system, the sample solution is injected into
the device for removal of extracellular ATP as pretreatment for
ATP-luminescence detection of intracellular microbial ATP.
Our pretreatment system consists of the microfluidic device, a
syringe infusion pump (KD Scientific, USA, www.kdscientific.
com) for sample injection, and the AFG 3102 Function
generator (Tektronix, USA, www.tek.com) (Fig. 2). The
function generator applies an AC current on the electrodes
of the device to generate DEP force.
Instruments for ATP-luminescence detection include an
electric circuit with photodiode BS520 (Sharp, USA, sharp-

Real-time detection of food-borne bacterial adenosine triphosphate

285

Fig. 1 Schematic diagram of


the extracellular ATP removal
process. Extracellular ATP in
food (i) is removed (ii) and only
intracellular ATP from
microorganisms is measured
(iii). Processes that occur in (ii)
are described below in detail.
a Microorganisms can be
captured on the electrodes by
DEP force while extracellular
ATP is not captured and passes
through. b Deionized water is
injected into the channel to
remove extracellular ATP,
leaving only microorganisms in
the channel. c Microorganisms
are lysed with ATP-releasing
reagent and the intracellular
ATP released only now enters
the ATP-luminescence reaction

world.com), NI USB-6229 BNC Data Acquisition and laptop


computer with LabVIEW 8.6 (National Instrument, USA,
www.ni.com). ATP-luminescence of intracellular ATP of
microorganisms is detected at the BS520 photodiode. The
circuit generates electric signals continuously and the electric
signals are proportional to light intensity detected at the
photodiode. The electric signals are changed to digital signals
with NI USB-6229 BNC Data Acquisition and LabVIEW 8.6
software reads the signals.

Fig. 2 Schematic diagram a and


picture b of the device. The
channel has a length from inlet
to outlet of 9 mm, height 50 m
and width 700 m. The chamber
in the middle of channel is
5,000 m long and 4,000 m
wide. The interdigitated
electrodes are 350 nm thick,
50 m wide and 50 m apart

E. coli cultured in LB broth (Alpha Biosciences, Inc.,


USA, www.alphabiosciences.com) were used for experiments. To lyse E. coli and release intracellular ATP, we
used an ATP-releasing reagent (Lucipac W, Kikkoman,
Japan, www.kikkoman.com). Adenosine triphosphate (Sigma, South Korea, www.sigma-aldrich.com) supplied extracellular ATP. ATP-luminescence assays were conducted
with D-Luciferin-Luciferase (Lucipac W, Kikkoman,
Japan, www.kikkoman.com).

286

Results and discussion


Capture of bacteria using dielectrophoresis
As shown in Fig. 3 E. coli cells are efficiently captured by
DEP force at 5 Vpp and 1 MHz of AC current. Further
measurement was required, however, to validate capture
in the working device, where cells are in flux. Hence,
yield of captured E. coli was measured by directly

H.-S. Moon et al.

counting the number of cells that the device could


actually capture at the DEP electrodes. We prepared E.
coli samples at 107 cellsmL1 in the deionized water. We
injected samples into the device at various flow rates
using the syringe pump, and for a preliminary test,
applied a DEP force at 5 V pp and 1 MHz. The
concentration of E. coli in the outflow was measured
using a hemocytometer. Capture yield is calculated by the
following equation:

Yield% 1  fconcentrationoutflow=concentrationinflowg  100

Figure 4 shows the capture yields at various flow rates.


As flow rate increases, the velocity of cells in the
horizontal direction increases. And therefore the duration
of time that cell experiences DEP force decreases, which
results in decreasing of yield. It is apparent that we can
get highest yield with slowest flow rate. However, for
that, we need more time to collect certain amount of
samples because of slow flow rate. Therefore we used the
flow rate of 10 Lmin1 which we can get 87.7% of
yield.
Measurement of bacterial ATP
To validate the device performance, we showed that (1) the
extracellular ATP is removed, and does not influence the
intracellular ATP determination, and (2) lysis of microorganisms completely releases the intracellular ATP for
detection in the bioluminescence reaction.

Fig. 3 Bacteria captured on the


electrodes with a DEP off,
and b DEP on. Without the DEP
force bacteria are dispersed in
the sample solution. With DEP
force, bacteria are attracted to
the strong electric field at the
edges of electrodes because
the DEP is positive.
c Cross-sectional view of
electric field is visualized using
COMSOL Multiphysics 3.2b.
Applied voltage was 5 Vpp,
1 MHz and the dimensions were
same as experimental ones

The E. coli concentration in a sample can be calculated


from the relationship between the output signal and cell
concentration displayed in a standard curve. For this, we
prepared standard E. coli samples at 103, 104, 105, 106, 10,7
and 108 cellsmL1 in deionized water. Each sample was
reacted with 100 L of D-luciferin-luciferase reagent
(3 mgmL1) and 200 L ATP releasing reagent (Lucipac
W, Kikkoman, Japan) added manually. The ATPluminescence of each sample was then measured by the
photodiode detector. To display the relationship between
the signal and E. coli concentration, we constructed a
standard curve using E. coli samples (Fig. 5). The signal
and bacterial concentration showed a near-linear relationship as shown in Fig. 5.
We tested the device holding constant the concentration
of either E. coli or ATP. If the device operates properly, the
intensity of light from the ATP-luminescence reaction after
the pretreatment process will not change against the

Real-time detection of food-borne bacterial adenosine triphosphate

287

Fig. 4 Yield of captured E. coli was measured by directly counting


the number of cells that the device could actually capture at the DEP
electrodes. As the flow rate increased, the yield decreased because the
duration of time that cell experiences DEP force decreased

extracellular ATP concentration and will be proportional to


the concentration of E. coli.
First, an E. coli constant sample (2107 cellmL1) and
ATP solutions (106, 107, 108, 109, 1010 M) were
prepared, then ATP at each concentration and the E. coli
standard were mixed. Each of these samples was injected
into the device at a 10 Lmin1 flow rate for 10 min, and
DEP force was applied. The extracellular ATP was removed
with deionized water (10 Lmin1, 10 min). The ATP
releasing reagent was injected (20 Lmin1, 10 min).
Samples of the outflow were reacted with 100 L of Dluciferin-luciferase reagent (3 mgmL1) and light intensity
was recorded. Figure 6a shows that the signals, with an
average value of about 1.1105, are constant against the
varying concentration of extracellular ATP. This result
indicates that the device effectively removes the extracellular ATP and only the intracellular ATP of E. coli takes
part in the ATP-luminescence reaction. If extracellular ATP
were not removed by the device, the signals would be
proportional to the concentration of ATP.
Second, an ATP solution (108 M) and samples of E. coli
at various concentrations (104, 105, 106, 107 cellsmL1)

Fig. 6 Signal output from samples containing a different concentrations of ATP and a constant bacterial concentration, and b different
concentrations of bacteria and constant concentration of ATP. Samples
were treated to remove extracellular ATP before the ATPluminescence reaction

were prepared in D.I. water. The ATP was added to E. coli at


each concentration, and the samples were injected into the
device (10 Lmin1, 10 min) with a DEP force applied (5 V,
1 MHz). Extracellular ATP was then removed with water
(10 Lmin1, 10 min). After 10 min, ATP releasing reagent
was introduced for 10 min at a flow rate 20 Lmin1. The
outflow was reacted with 100 L of D-luciferin-luciferase
reagent (3 mgmL1, Kikkoman, Japan) and the light
intensity was measured. This output signal was proportional
to the concentration of E. coli as shown in Fig. 6b. The
signal values at E. coli concentrations of 104 and 105
cellsmL1 are 2.1108 and 1.8107, respectively, significantly lower than 7.1106, which corresponds to 1.0
108 M ATP. That means the ATP luminescence reactions are
not influenced by extracellular ATP. If extracellular ATP was
not removed by the treatment, the signal slope would reach a
plateau at a lower E. coli concentration.

Conclusion
Fig. 5 Photodiode signal output as the concentration of bacteria is
varied. The signal and bacterial concentration have a near-linear

relationship. y 1:84E  12x 6:84E  10; R2 0:98

We constructed a microfluidic pretreatment device which


removes extracellular ATP and permits accurate detection
of intracellular microbial ATP. The device uses dielectro-

288

phoretic force to capture microorganisms and measures the


ATP content by ATP-luminescence. With conventional softlithography and metal etching processes, we constructed a
microfluidic device which can generate DEP force. To
validate the device, we performed experiments and found
that the yield of capture by DEP force at a 10 Lmin1
flow rate was 87.7%. And the removal of extracellular ATP
from samples containing extracellular ATP mixed with E.
coli was confirmed by experiments.
We expect the shelf-life of the microfabricated device to
be virtually infinite because Au electrode, glass wafer and
PDMS, those we used so far, are inert materials and they do
not need any biochemical modification. Unless sample
includes rather large food particles which can block the
electrodes or channel, we believe our device can treat real
samples with minimal sample volume, cost effectiveness,
rapidity and reliability.
Acknowledgement This work was supported by grants from the Korea
Institute of Environmental Science and Technology (Grant 101-082-035)
and facilities were kindly provided by the National Core Research Center
for Nanomedical Technology (R15-2004-024-00000-0) and ICBIN of the
Seoul R&BD program (Grant no. 10816).

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