Analyte derivatives and immunogens were prepared for the target substances. This phase was followed by immunising appropriate numbers of antibody producing units. The maturation of the immune response was followed by specific ELISA monitoring tests. Antibodies to most of the target analytes were successfully prepared with the exception of the difficult group (nonyl phenol. MTBE, glyphosate). The quality of antibodies varied across the panel, influenced by the structure of individual analytes. Competitive ELISA and parameters pf assay response were developed for the target substances using a second set of analyte derivatives (critical part of immunochemistry WP) and antigen-coated assay methods. Purified antibodies were isolated using individual affinity chromatography procedure. This involved the preparation of column material and optimisation of elution systems. The provision of preparative quantities of pure antibodies was critical to the success of the project. Deliverables included: pure antibodies, analyte derivatives, immunoassay methods, cross reaction data.
An integrated optical chip is being developed by the ORC, which will allow 32 simultaneous fluorescence-based immunoassays to be conducted with high sensitivity in a compact instrument. Optical wave guides are defined at the surface of a glass “chip” using the fabrication techniques familiar from microelectronics. These wave guides conduct light around the surface of the chip, in an integrated optical circuit, to the 32 sites where the surface is chemically modified for specificity to a particular analyte. Light is introduced from a semiconductor laser diode and wave guides are optimised at the sensing sites to yield efficient excitation of surface fluorescence using the evanescent fields. The final chip size is approximately 15mmx68mm, and the fluorescent patch sizes are approximately 0.2mmx1.5mm. Prototype chips have been fabricated and are presently being tested without permanently attached fibre pigtails. Applications of this result will be in environmental monitoring, medical diagnostics and biotechnology. The applications are not for screening very large numbers of compounds in high concentrations, but fairly low numbers of compounds in very low concentrations. End-users will include instrument manufacturers and scientific researchers who wish to analyse small volumes of analyte for multiple compounds with good stability and repeatability and low detection limit. The main innovative features are the wave guide design leading to high sensitivity, the massfabrication technology employed for the chips, which will help keep unit costs down, and the robust connection to instrumentation which is expected to lead to greater reliability and ease of use and to reduced detection limit through improved stability. These chips are being developed for fluorescence-based measurements but may be applied to other surface scattering or luminescence techniques, such as Raman spectroscopy.
This module covered the design and development of the microfluidics system and the assembly of the multi-channel, automated TIRF-based immunosensor instrument. The unit was designed to be small enough to be carried to a test river site where it could be installed to provide continuous surveillance of pollution levels for up to 32 different chemical compounds. The first stage in this process required agreement on the technical specification of the instrument and this involved extensive discussions between both ‘user’ and ‘supplier’ partners within the Consortium, concluding with the preparation of a technical specification manual, which defined the design parameters for the major components of the system. The result of this collaboration was the fabrication, testing and development of a 32-channel analyser, consisting of integrated electronics, and optics and fluidic modules. The electronics modules consisted of pumps, valves, power and laser supplies housed in a metal casing. The metal casing provided Rf shielding for low noise linear amplification circuits that interfaced with a 32 photodiode array, which were housed in a separate light-tight metal compartment. The microfluidics component consisted of a PMMA embossed flow chamber, with a critical chamber dimension of approximately 35µm depth, covering an array of 32 sensor zones, each zone being 1.5mm long and 300µm wide, on a fibre pigtailed TIRF chip. Tests showed that the chamber structure maintained poiseuille fluid flow across the sensor region and it could therefore deliver binding assay results with good repeatability. The photodetection system consisted of an array of highly polished optical fibres in close contact with the sensor chip at one end and the photodiode array at the other. The amplified signals from the photodiodes were transmitted to the measurement control and data acquisition system through individually shielded outputs. The analyser underwent a commissioning and bench testing regime by CRL, Southampton University, Siemens and Tuebingen University and the fully integrated instrument, including the computer interface and autosampler modules, will now be permanently attached to Tuebingen University, where it will continue to be tested and developed. A second instrument will undergo field testing by the other ‘user’ partners. Specification documentation and a summary operating instructions manual have been produced and supplied to the ‘users’. These documents are intended to support future opportunities to supply instruments to customers on a commercial basis.