Microfluorimeter with disposable polymer chip for detection of coeliac disease toxic gliadin (1)
Coeliac disease is not an allergy; it is an auto-immune disease, which means that the body produces antibodies that attack its own tissues. The new benchmark states that foods labelled ‘‘gluten-free’’ may not contain wheat, rye, oats or barley and the gluten level may not exceed 20 ppm (parts per million). Foods that have been processed to reduce gluten content to a level between 20–100 ppm cannot be labelled gluten-free but may be called ‘‘low gluten’’ or ‘‘reduced gluten’’.
The concept of the chip-based multi-parameter analysis system includes a novel integrated reflector arrangement for the detection of fluorescence. For the fluorescence excitation, the beam is guided through the detection cell directly to the detector using 45 -tilted mirrors located in the direction of the beam. Fluorescence detection is achieved by a mirror arrangement oriented in parallel to the detection cell which collects the emitted light and concentrates it onto the detector.
The micro-fluidic analytical chip integrated with the fluorescence detection unit has five separate channels for fluorescence measurement.
Competition ELISA: colorimetric detection. The prepared biotinylated gliadin (500 ng/mL, 10 nM) was immobilised by incubation of 50 mL in each well of a streptavidin-coated microtitre plate for 1 h at 37 C in PBS-Tween (0.01 M, pH 7.4). At the same time, 60 uL of a range of gliadin concentrations (from 18000 to 0 ng/mL), 60 mL of monoclonal anti-gliadin antibody diluted (1 : 3000) and 60 uL horse radish peroxidase- labelled rabbit anti-mouse IgG, (HRP-anti-IgG, 1 : 3000) were added to an Eppendorf tube and allowed to pre-incubate for the same hour at 37 C. Following this first incubation, the plate was thoroughly washed with PBS-Tween buffer, and 50 mL of the pre-incubated solution was introduced into each well. After 10 min incubation at 25 C, the plate was washed 3 times with PBS-Tween and 50 mL of tetramethylbenzidine (TMB) substrate was added to each well. Color development was monitored at 650 nm, stopped after 20 min using 0.1 M H2SO4, and finally measured at 450nm.
The limit of detection (LOD), IC50 and hillslope obtained and as can be seen, the LOD obtained are 4.8, 0.7 and 0.1 ng/mL gliadin for the HRP, ALP and FITC labels, respectively.
The detection limit obtained was similar to that obtained using the standard laboratory spectro- fluorimeter, with an LOD of 4.1 ng/mL being obtained. The reproducibility of the system was evaluated using 50 ng/mL of gliadin by carrying out the fluoroimmunoassay in each of the 5 channels of the microfluorimeter.
An excellent correlation with the colorimetric ELISA is obtained, demonstrating the usefulness of the developed microfluorimeter for in situ analysis of gliadin in raw materials and as a control of contamination along the food production line, for use by food industries.
