Resultado final
An immunosensor using resonant mass transduction with a quartz crystal microbalance for the detection of gliadins, exploiting the monoclonal antibodies developed within the consortium. The immuno-sensor is designed to be lab based and re-usable. Detection limits obtained with direct capture of an antibody immobilised via a monolayer self-assembled on the gold electrode of a quartz crystal were not sensitive enough and thus an indirect sandwich immuno-sensor has been developed.
In terms of commercial interest, this immuno-sensing platform is not designed for use in situ, and is not disposable and nature and has been specifically designed to be laboratory based and an instrument has also been developed. The SME who developed the instrument have an interest to exploit the instrument themselves and are open to collaboration to commercialise functionalised quartz crystals specific for gliadin detection.
Enzyme linked immunosorbent assays (ELISA) for the detection of coeliac disease toxic gliadins. ELISAs have been developed using monoclonal antibodies produced against the putative immunodominant epitope as well as alpha gliadin 31-45. Sandwich and competitive ELISAs have been developed. These assays have been completely validated using spiked samples, are highly reproducible and capable of detecting ppb levels of wheat, rye, barely and oat prolamins, with absolutely no cross reactivity to rice and maize.
An aptasensor using resonant mass transduction with a quartz crystal microbalance for the detection of gliadins, exploiting the gliadin aptamer developed within the consortium is proposed for exploitation. The aptasensor is designed to be lab based and re-usable. Detection limits obtained with direct capture of an aptamer immobilised via a monolayer self-assembled on the gold electrode of a quartz crystal reached ppm detection levels.
In terms of commercial interest, this aptasensing platform is not designed for use in situ, and is not disposable in nature and has been specifically designed to be laboratory based and an instrument has also been developed. The SME who developed the instrument have an interest to exploit the instrument themselves and are open to collaboration to commercialise aptamer functionalised quartz crystals specific for gliadin detection.
An ELISA for the detection of hydrolysate forms of coeliac disease toxic prolamins has been developed for the application to glucose syrups (dextrose, maltodextrins) and beer. The ELISA detects ppb levels and the use of peptide-tryptic digests and standard gliadin as coating antigen and standard have been compared and the use of the peptide-tryptic seen to be far superior. The ELISA has been validated using spiked samples and has been applied to the analysis of real samples and has been demonstrated to be highly reproducible.
A microsystem module for optical detection has been developed and demonstrated for fluorescence detection. The optical detection chip was designed to obtain 5 parallel cuvettes for the dilution series of the metering chip, which can be operated by a sledge on a linear translation stage holding one light source for the excitation and two different photodiodes for detection; one for an UV/Vis measurement and one fluorescence detector. The UV/Vis detector can be used twice, for absorption measurements to detect the concentration of the dye, but also for the positioning of the sledge in the centre of the channel. The performance of the optical detection chip has been tested for transmission measurements in the UV/Vis range and for fluorescence measurements.
The sensitivity for transmission measurements was tested with methylumbelliferone at 370nm. Methylumbelliferone is a typical product of ELISA assays when the enzyme alkaline phosphatase is linked to the secondary antibody. The sensitivity is comparable or even better to standard laboratory photometers. Experiments with 5,6 FAM were carried out to test the sensitivity for fluorescence measurements. In these experiments samples of different concentrations of a fluorescence dye were filled into the cuvette channel. This is comparable to an assay in which an enzyme is conjugated to the secondary antibody. This enzyme produces a fluorescing dye which is released into the channel volume. A good correlation was obtained with a sensitivity of ca. 20nM which is suitable for the detection of gluten.
An ELISA has been developed for the detection of HMW glutenins. The developed ELISA is based on a competitive assay and the use of isolated (via sequential extraction) HMW glutenins and recombinant HMW glutenins as coating antigen as well as standards has been compared and both function equally. The assay has been validated using spiked samples and is very reproducible. Sub ppb detection limits have been achieved.
The developed assay has been applied to real samples where detection of both gliadins and glutenins has been carried out and it is clearly demonstrated that the extrapolation of [total gluten] = [gliadin x 2] is incorrect and with the new knowledge that HMW glutenins are as, if not more, toxic than gliadins, it will be necessary to measure both gliadin and glutenin content to get a true idea of total gluten content.
An impedimetric aptasensor has been developed for the direct detection of gliadin in foodstuffs exploiting an aptamer specific for gliadin that was developed within the consortium. The aptasensor is based on the thilated apatmer being immobilised on a gold electrode and is capable of ppm detection limits. Further developmental work is required to fully validate the aptasensor and efforts are required to reduce the detection limit. The aptasensor can be regenerated and is compatible with the extraction buffer developed within the consortium.
A universal protocol for the combined extraction of gliadins and glutenins from both raw and cooked foodstuffs. The extraction process improves significantly on the state of the art, dramatically reducing the required extraction time from 3 hours to 10 minutes, avoiding the use of noxious and odorous reagents and reducing the volume of required extraction solvent. The extraction protocol is completely compatible with ELISA and biosensor formats.
A microsystem that integrates the modules of extraction, metering, dilution, reagent delivery and detection has been demonstrated as a laboratory prototype. The individual micromodules have been realised in single polymer chips. To obtain a complete system these chips have to be assembled on a fluidic platform. The chip-based-lab platform developed at IMM was specially designed for this purpose. A metal plate with metal frames can house the fluidic chips in any desired combination. Interfacing chips are used to get the fluidic contact between the chips. Active flow control components have been described in previous abstracts.
These are:
- A syringe pump. This pump is connected to several inlet ports addressed by external valves to avoid a too deep pressure drop. This could be obtained if all chips would be addressed by only one single port.
- Light barriers that monitor the arrival of fluids at specific spots of the chip to indicate the right time to switch valves.
- Valves. Internal valves follow one single principle. Different versions vary only by the design of the slits in the rubber disk. All valves are actuated by electric stepper motors. Their position is monitored by common forked light barriers.
All components are controlled by an electronic compartment which was designed also in a modular way reflecting the same flexibility that was gained on the fluidic side with the chip-based-lab platform and its standardised fluidic chips.
The assembly of a complete system was realised with different aspects.
-·Automatic switching of the valves
-·Automatic monitoring of the plug position
-·Automatic operation of the extraction motor
-·Automatic operation of the centrifuge including centrifugation and the filling process
-·Automatic detection signal read out
-·Interface to a fluidic actuation system with pumps and external valves
-·Control of the progress by an electronic system
The functionality of single fluidic chips has been tested successfully. In the actual state, combinations of several chips were tested. In detail this has been
-·Extraction and centrifuge
-·Dilution and mixing
-·Dilution, mixing and distribution
-·Metering and distribution
-·Metering, distribution, ELISA and detection
-·Storage (ELISA), Metering, distribution, ELISA and detection
Since the realisation of a functional dilution chip was achieved very late at the end of the project, as well as the complete actuation, monitoring and electronic control, only first tests of the complete system were performed. Primary results showed that the system is working in principle but that there is extra effort needed to obtain a robust system for the measurement of real samples.
An aptamer has been selected using fluoro-modified RNA against a combination of coeliac disease toxic peptides including the putative immunodominant epitope as well as against the whole gliadin protein. The selected aptamer has nanomolar affinity and has been applied to aptasensor development.
A semi-automated microsystem for the extraction of gliadins and glutenins from raw and processed foodstuffs had been developed based on the universal extraction protocol developed within the consortium. This extraction protocol was transferred into 3 single modules:
-On-chip storage of extraction buffer
- Extraction module
-·Centrifuge
Besides the storage of the extraction buffer this chip provides the interface to the actuation system of the instrument. Here one syringe pump is required and one external valve to apply the pressure to these 3 modules. The extraction has to be pre-filled at the production site, no filling is needed during the analysis.
The extraction module imitates the vigorous mixing transferred into a microsystem. Mainly, it is a flexible silicon tube in which the sample is filled in. Via a valve the extraction buffer is filled into the tube. A bar with two wheels is lowered down to the tube and rotated by an electric motor forming a peristaltic pump. Underneath the tube a heating coil is mounted to obtain temperatures of up to 90°C for the extraction of processed food. Disposable components are the blue polymer block in which the valve is integrated, the silicon tube and the tube connectors between tube and chip. All other components will not be in contact with the sample, will not be contaminated, and will be a re-usable component of the instrument.
An optical biosensor exploiting surface plasmon resonance detection has been developed for the detection of gliadin, using an aptamer (developed within the consortium) as the biocomponent. The developed aptasensor can detect down to low ppm levels of gliadin, can be regenerated up to 20 times and is compatible with the extraction buffer developed within the consortium. Considerable work is still required to improve the detection limit of the aptasensor so as to be applicable to gluten free foods where a detection limit of 10ppm is required.
For the quantitative determination of the gluten concentration using biosensors several consecutive steps are necessary. First a dilution series has to be generated. The sample has to be mixed with immobilised antibodies in the real detection module and all reagents that belong to the assay have to be stored, metered and distributed to the detection zone. A signal is generated at the end that will be detected by optical or electrochemical detectors that are partially integrated into the chip.
The generation of a dilution series is required since the concentration range of gluten can span several orders of magnitude. Thus, a dilution seems to be mandatory to obtain at least one dilution with an acceptable size of the detector signal. With this system 4 consecutive 1:3.16 dilutions are generated enable measurements of the original concentration down to a 1:100 dilution. For this purpose, the sample coming from the centrifuge is metered twice. Using the turning valve already introduced at the part of the extraction module, first a metering loop is filled followed by a channel in which a light barrier stops the sample at a precise position. Turning the valve the first part (metered by the light barrier) is directly transported to the detection chip. The sample that is stored in the metering loop is mixed with a pre-metered volume of dilution buffer and sent to a mixing chip. The mixing chip provides only long meandering channels, in which the two fluids will mix. The mixed and diluted sample is transported back to the dilution chip in which the same procedure like for the first dilution is repeated three times.
The supply of the reagents is performed by a combination of three chips. The first one provides the stored liquids. Since they have to be used several times 5x5 times a washing buffer, 5x1 the secondary antibody and 5x1 the substrate, the complete volume of a chip is needed. This chip also provides the interfaces to external valves and the syringe pump. These additional pressure inlets were chosen to avoid too long channel systems in which a control of the pressure and fluidic flow becomes more and more complicated.
The metering chip takes advantage of the same arrangement of the turning valve and metering loops that have been already used in the dilution chip. The metering loop is emptied and its content transported to the distribution chip. Here, the metered reagents are distributed by 5 turning valves to the detection zones. The design of the channel is chosen in a way that in this chip samples never use the same channel. All reagents will not use the same channel before the valves are reached. This should minimize contamination of the reagents by molecules that stick unspecificly to the channel walls. The most critical step is the delivery of the secondary antibody. If this reagent binds unspecificly to the channel wall subsequently delivered substrate could also react with these antibodies disturbing the signal significantly. After surface modification leading to more hydrophilic surfaces this source of errors could be reduced significantly.
Recombinant gliadin and HMW glutenin proteins have been expressed in tobacco plants. These proteins could be used as standards for ELISA development. Additionally they could be used as coating proteins or labelled and used as competitors for ELISAs.
Monoclonal antibodies against the coeliac disease putative immunodominant epitope as well as other minor epitopes. These antibodies have been characterised and are capable of low ppb detection limits. They do not cross react with rice or maize, and do cross react with rye, barely and to a smaller extent oats.
Monoclonal antibodies against HMW glutenins. These have been developed using Dx5 and Dy10 subunits as and are also capable of ppb detection limits. They do not cross react with gliadin.
A microsystem module for electrochemical detection has been developed. The measurement of the gluten concentration by an electrochemical sensor requires the implementation of electrode pairs into the detection chip. The reported approach provides a sandwich of 5 gold working electrodes with immobilised antibodies and an Ag/AgCl counter/reference electrode, whose surface is significantly larger than those of the working electrode.
An array of 5 gold working electrodes is sputtered on a thin glass slide with titanium as intermediate layer. The usage of glass as a substrate has two important advantages, glass can be cleaned under harsher conditions compared to polymers and sputtered gold on glass substrates are known as very stable. An electrical interface is provided by small gold strips leading from the electrode spots to the edge of the glass slide. This glass slide is slipped into a foil connector which is commercially available and guarantees a close contact of the electrodes with the connections to the instrument. On top of the gold electrode array double sided adhesive tape is bonded. In this tape slits are cut by laser ablation. These slits form the fluidic channels inside of the detection sandwich. On top a second slide is fixed that is completely covered by an Ag/AgCl-layer that serves as counter and reference electrode. Holes in this layer and holes at the same position of a second layer of double sided adhesive tape build up the fluidic interface between the sandwich and a the detection chip which is part of the fluidic platform. While the working electrodes are connected via a foil connector is the counter/reference electrode connected by a pogopin which is fixed underneath the chip level.
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