Mycotoxins are health hazardous metabolites produced by fungi growing onfoodstuffs or animal feed. Upon exposure they impact on the productivity oflivestock including diary products. As the result of livestock feeding on conta-minated commodities, mycotoxins carry over to the human food chain viameat or other animal products. This poses a high risk to human health as someof the most common mycotoxins are carcinogenic, genotoxic, or target the kid-ney or liver. It is now well established that acute mycotoxicoses (the diseasescaused by mycotoxins) have been responsible for major epidemics in man andanimals.
Since mycotoxins are robust, small molecules that are not easily destroyed bytemperature treatments occurring during cooking, baking or freezing processesand even fermentation, it is necessary to avoid usage of contaminated foodand feed upfront. However, the presence of mycotoxins is not detectable byvisual screening due to the fact that high amounts of toxins may still exist evenafter removal or destruction of the generating organisms. Hence availability ofsensitive, accurate and affordable tests for the detection and quantisation ofmycotoxin in feeds is of great interest for the care of health.
Here, we introduce an automated Lab-on-
a-Chip system for reliable quantitative
testing of mycotoxin contamination of
animal feed (cf. figure 1) developed in theframework of the EU-funded project micro-FLUID [1].
Employment of microfluidic analysis tech-niques facilitates miniaturisation, allowingfor the use of small quantities of reagents
to perform assays. This reduces costs and, avoids the handling of large amountsof dangerous toxins. The portability of the system enables in-field analysis andprovides a significant improvement in the prevention and control of the conta-mination. The instrumental reading of the optical intensities ensures a reliableand quantitative interpretation of the result.
The LOC system is based on a disposable microfluidic polymer chip (cf. Fig 1)equipped with an optical immunoassay technique for detection. Due to smallchannel dimensions, substantially reduced incubation times are achievedcompared to these typically required in respective commercial assays carriedout in 96-well plates. Furthermore, our fully automated system allows for
direct detection of mycotoxins in raw samples [2,3]. In the extraction processrecovery rates higher than 80 % have been found. After extraction, the analyteis automatically transferred to microfluidic channels where immunoassay-based fluorescence detection takes place. The concept of the implementedELISA is illustrated in the following figures:
Step 1: Extraction of analyte (here: Aflatoxin B1)in attached unit (large red cycle) or external.Lyophilised reagents (orange channels), surfaceimmobilised antigens (green channels) are storedon-chip. Containers holding buffer or extractingagent (coloured circles) are attached to the chip.
Fig. 1: Disposable polymer chip with attachedextraction unit inserted into the fully automateddevice incl. actuation and detection modules.
Lab-on-a-Chip System for Detection
of Mycotoxins in Animal Feed
Introduction
System Design and Results
References Acknowledgement
Contact: [email protected]
www.imm-mainz.de http://www.agrolabo.it
S. Brunklaus1, V. Stein1, M. Jakubowski1, M. Ritzi-Lehnert1, K.S. Drese1, R. Colasanto2, P. Poletti2
1Institut für Mikrotechnik Mainz GmbH, Carl-Zeiss-Str. 18-20, 55129 Mainz, Germany2Agrolabo S.p.A., Via Masero 59, 10010 Scarmagno (TO), Italy
2606
2011
Step 2: Metering of analyte (red), dilution buffer(yellow) and buffer (magenta) for the re-dissolv-ing of lyophilised calibrators and/or conjugate (= antibody-HRP) in specifically designed meteringloops. Subsequently, reagents pre-stored in chan-nels are re-dissolved by bidirectional movementsof fluid plugs.
0.3
0.35
0.4
0.45
0.5
0.55
0 10 20 30 40
time in min
UF
in V
1 100
50
100
150
200
250
300
350
(UF -
UB)
in m
V
H2O2 concentration in mmol/l
Step 4: Incubation of analyte/ conjugate mix and
calibrator/ conjugate solutions (magenta) in de-tection channels above immobilised antigens.Excess antibodies which are not bound to an anti-gen stemming from calibrator or analyte will bindto the antigens immobilised previously to thechannel surface.
Step 5: Metering of washing buffer and subsequentwashing of detection channels. Non-immobilisedduplexes of conjugate and antigens are removedto waste containers behind the detection area.
Step 6: Metering of substrate and subsequentincubation of substrate in detection channels.
During the incubation a time the substrate (here:Amplex Ultrared) is converted to its fluorescingform. Thus, the resulting fluorescence signal isinvers to the mycotoxin concentration present inthe analyte.
Step 7: Read out of fluorescence signal by a large area photodiode below detection channel.
[1] http://www.ifn.cnr.it/microfluid
[2] Further development of device covered by patent application DE102008042581.8-41
[3] M. Jakubowski, diploma thesis, Institut für Mikrotechnik Mainz, University of Applied Sciences Bingen, 2009
This work has been carried out with financial support from the EuropeanCommission, Seventh Framework Programme, grant agreement no. 224205,project ‘microFLUID’.
The introduced mycotoxin detectionsystem allows for automated directdetection of mycotoxins at produc-
tion sites by moderately trained per-
sonnel, thereby providing a highlyeffective means to minimize myco-toxin contamination of livestockduring the food production chain.
The principle design of the disposablemicrofluidic chip is highly compatible
with cost-effective, large-scale fabri-
cation techniques such as injectionmoulding. Most importantly, our LOCsystem can be readily equipped forsimultaneous detection of all relevantmycotoxins in animal feed when ini-tially implemented calibrators are ex-changed by different mycotoxins.
Fig 2: Dynamic measurement of fluores-cence signal UF during aflatoxin B1 assay(different symbols for each channel).
Fig 3: Fluorescence signal UF in AmplexUltrared/peroxide assay (background signalUB subtracted).
