CSEM Scientific and Technical Report 2008

Lab-on-a-Chip for Analysis and Diagnostics: Application to Automated Detection of
Antibiotics in Milk
G. Suárez, S. Pasche, R. Ischer, G. Voirin, S. Berchtold, N. Schmid, J. Auerswald
An integrated optical biosensor has been combined with microfluidics in order to obtain a multianalyte automated sensing system. This lab-on-achip
approach was tested for the daily control of milk at the dairy farm for antibiotic contaminants.
In industrialized countries, food safety is subject to strict
legislation that regulates the presence of contaminants in food.
Antibiotics can often be found in milk due to their routine use
for live-stock treatment in case of bacterial infection and/or for
prophylactic purposes. Particularly, four antibiotic families
were identified for their probability of occurrence in milk:
sulfonamides, fluoroquinolones, β-lactams and tetracyclines.
Their abundance in the human diet may represent a serious
problem by inducing bacterial resistance to these antibiotics.
Consequently, maximum residue limits (MRL) of 4 ppb for
β-lactams and 100 ppb for the other antibiotics have been
established for milk. Conventional detection methods, mainly
chromatography coupled to mass spectroscopy, offer high
screening capability but are costly and require a laboratory
environment. On the other hand, fast dipstick tests are
available but currently do not allow detecting more than two
antibiotic families simultaneously. In this work, the
development of a cheap and disposable lab-on-a-chip device,
which allows simultaneous detection of four antibiotic families
in less than ten minutes, is reported.
The detection principle is based on a solid-phase
immunoassay which is monitored with the wavelength
interrogated optical sensing (WIOS) technology (developed at
CSEM and commercialized by Dynetix). Each sensing chip
comprises eight individual sensing regions that are made
sensitive to different antibiotic families through
functionalization with specific capture biomolecules. As shown
in Figure 1, the sensing chip is covered with a fluidic chamber
and inserted into a microfluidic cartridge that contains the
reagents in reservoirs. The microfluidic cartridge
(10x4x0.7 cm3 ) is made of plastic (PMMA) using micromilling
and thermobonding to create open channels.
Figure 1: Schematic representation of the microfluidic cartridge
During the immunoassay, sequential delivery of the different
solutions contained in the microfluidic cartridge reservoirs is
achieved using the coordinated actuation of a pump and a
valve. The cartridge was designed to avoid air bubbles. Waste
reservoirs limit the risk of contamination by storing all residual
liquid within the cartridge. The main actions for lab-on-a-chip
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detection of multiple antibiotics in a milk sample are
summarized in Figure 2.
a) b)
c) d)
Figure 2: Sequence of operations for lab-on-a-chip multiplexed
detection of antibiotics in milk: insert cartridge (a), add 40 µl of milk
into the vial (b); plug the vial into the instrument (c); run the software
and read the results (d).
Milk samples spiked with MRL concentrations of sulfapyridine,
ciprofloxacin, ampicillin and oxytetracycline – representative of
the four antibiotic families – were tested and compared with
antibiotic-free milk. The WIOS responses obtained after a
10 min assay show signal inhibition in the presence of
antibiotics (Table 1), demonstrating clear discrimination
between positive (below MRL) and negative (at MRL)
concentrations for the four antibiotics.
Table 1: WIOS signals inhibition at MRL for different antibiotics
(sulfapyridine, ciprofloxacine, ampicillin and oxytetracycline)
Antibiotic Sulfa Cipro. Amp. Oxytet.
MRL (ppb) 100 100 4 100
Inhibition at
MRL
77 % 74 % 26 % 62 %
The reliability of the lab-on-a-chip detection system was
successfully verified with a validation test carried out on a
series of six blind milk samples provided by Nestlé leading to
a test accuracy of 95%.
The disposable lab-on-a-chip device allows simultaneous
detection of four antibiotic families in milk with a fast and
simple approach which requires neither a laboratory
environment, nor skilled personnel. This device is therefore
suitable for use at any milk collection point such as the dairy
farm or the collecting truck.
This work was funded by the Competence Centre for
Materials Science and Technology (CCMX) of the ETH-Board,
Switzerland. CSEM thanks them for their support.