Beruflich Dokumente
Kultur Dokumente
DOI 10.1007/s00604-010-0370-9
ORIGINAL PAPER
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
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
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-
285
286
287
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
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
288
References
1. Hilleman M (2002) Overview: cause and prevention in biowarfare
and bioterrorism. Vaccine 20:30553067
2. Bogdanovic J, Koets M, Sander I, Wouters I, Meijster T, Heederik
D, van Amerongen A, Doekes G (2006) Rapid detection of fungal
-amylase in the work environment with a lateral flow immunoassay. JACI 118:11571163
3. Pyankov O, Agranovski I, Pyankova O, Mokhonova E, Mokhonov
V, Safatov A, Khromykh A (2007) Using a bioaerosol personal
sampler in combination with real-time PCR analysis for rapid
detection of airborne viruses. Environ Microbiol 9:9921000
4. Sengupta A, Laucks M, Dildine N, Drapala E, Davis E (2005)
Bioaerosol characterization by surface-enhanced Raman spectroscopy
(SERS). J Aerosol Sci 36:651664