Deliverables SAM based surface chemistry for biosensor development A common bottleneck in the development of biosensors is the insufficient stability and reproducibility of the biosensor interface, i.e. the interface between the inorganic transducer surface and the biological affinity elements, in the different environments of their practical applications. In addition, the increasing miniaturisation of biosensor transducers (and thus of their active areas) and the need for a higher sensitivity impose more severe demands upon the process of coupling biomolecules to transducer surfaces. Therefore, controlled thin film structures should be prepared in which the bio-affinity elements may be arranged and addressed in reproducible and controlled geometric surroundings. One of the most promising methodologies in order to achieve this is the use of Self-Assembled Monolayers (SAMs). Since the transducer surfaces of the affinity-based biosensors (e.g. immunosensors) developed at IMEC are composed of metals and/or oxide materials, our surface chemistry research is focussing on the covalent attachment of antibodies on metal (mainly Au) and on oxide surfaces (mainly SiO2 and Ta2O5). In particular, our approach to realise bio-reactive gold surfaces is based on the deposition of mixed SAMs of thiol compounds on clean gold. In such a strategy, one type of thiol in the mixed SAM carries a functional group to attach the receptor molecules, i.e. the antibodies, and another type of thiol compound allows for the minimisation of the non-specific adsorption of undesired bio-species. This greatly contributes to a solely specific interaction of the analyte with the immobilised antibodies. Camel antibodies bio-engineered agaisnt PSA Multiple (#25) single-domain antibody fragments derived from dromedary heavy-chain antibodies, and with specificity for PSA have been isolated. The kinetic rate constants of these single-domain antibody fragments, and their equilibrium constants for the antigen have been determined. The best binder 'cAb-PSA-N7' having an affinity for PSA in the pM range was re-cloned in a bacterial expression vector. The PSA binder can be obtained in mg quantities from one litre culture, and is very stable. It has been used for immobilisation on the biosensor layers, either by direct coupling or via fusion with the S-layer protein. To increase the PSA-capturing efficacy of the cAb-PSA-N7 we introduced 3 extra lysine residues at the C-end of the protein allowing a directional chemical coupling to carboxylate groups. In addition a bivalent construct was generated, however, its expression in bacterial fermentation remained low. The recombinant cAb-PSA-N7 could detect down to 1 ng PSA/ml in a Biacore-3000 biosensor. The binder is more stable than any other antibody fragment, which is an advantage and prerequisite for economical use of a biosensor chip. Molecular glue for bonding technology Bonding is one key technology in the fabrication of micro-chips. Anodic bonding is for example used to bond silicon and glass wafers; though, this technique is expensive and needs high voltage (400 � 1000 V) and elevated temperature (300 degrees C). Fusion bonding is also used to join quartz and Pyrex materials; quartz wafer is suitable to optical analysis, however, the technique requires more than 1000 degrees C and takes about one day to bond two wafers. An alternative is HF and water glass bonding, which are used to bond glass and silicon wafers for MEMS and µ-TAS respectively; handling HF is very dangerous, and the water glass is sodium rich, which is not applicable for chemical analysis. Glue bonding is also suitable for the µ-TAS; this technique brings macroscopic amount of extra material (silicone, epoxy resin) and requires curing (with temperature, UV light,�) to bond the two wafers. Approach: Our approach is based on the use of simple commercial molecules and has the following advantages: - Very little amount (2µl)of material necessary; - Very easy deposition of the 'glue' monolayer; - Does not need to apply pressure; - Glue layer thickness is in the range of few nm (less than 10 nm); - Perfect for flat/smooth surfaces; - Does only improve with time, by nature of the chemistry; - Very resistive to humidity; - Works at room T; T increase only speeds up a first bonding phase; a key factor for biochips; - Could be used for silicon wafers, or other flat metal oxide surfaces; - May be adequate for "stamp" deposition technique. LISE is still evaluating the possibility to apply for a patent. In the meantime, no more information is given. Microfluidics of the PAMELA biochip and SPR integration The integration and assembly of microTAS based microfluidics with the AW and SPR sensor systems was the general role of MESA+ in the PAMELA project. During the project a dramatic change came up after the design and realization of the initial single point or few-points measurement chips. Spotting of ligands using TOPSPOT microfluidics and the invention of the address flow principle lead to a dramatic new insight in the performance of chip technology, leading to "fingerprint" analysis. The integration with the new imaging SPR instrument from HBM was succesful and at MESA+ ultra low sensitivities were achieved of 0.15 ng/ml PSA in a 3% protein buffer system. Also the serum sample measurements, although limited in numbers, were successful. MESA+ will continue in the technological related area of "proteomics on a chip" and understands that without the PAMELA project it never had such a progress in the past few years. iSPR instrument The Pamela project ended with a functional model of the iSPR instrument. After the Pamela project was finished, two prototypes of the commercial iSPR instrument were built. After their extensive evaluation, the production of the final instrument was started. Currently, 10 months after the Pamela project was finished, a production line has been realised and the first instruments are assembled and sold. The commercial iSPR instrument is a versatile research instrument with an outstanding performance. The instrument consists of the iSPR unit and a dedicated computer on which the iSPR data acquisition software runs and that controls the iSPR instrument. The instrument uses two displays, one for data in and output and the other for image monitoring. The instrument includes a x-y-z robot that allows unattended operation. The sample compartment of the instrument is compatible with common multi-well plate technology and up to 1536 samples can be stored inside the instrument. The instrument uses the Kretschmann configuration and can be operated with scanning or fixed optics. The optical resolution of the instrument is less than 30 micron, which allows the study of 15.000 interactions simultaneously. The angle range is 22 degrees of which 4 degrees are dynamic. It is expected that more than 250 instruments will be sold worldwide during the next 7 years and that the breakeven point is reached in 2006. Clinical evaluation and limited market research The role of Aventis in this project was the clinical evaluation of the biosensor and using its network of physicians/specialists to assess the need and criteria for a new method for the analysis of free and total PSA analysis. Urologists are requesting the total PSA and free PSA analysis for their patients about 1200 times (mean) per year per specialist. This analysis is both requested for diagnosis as monitoring of the disease. Although they indicated to be satisfied with the current methods, the major part (82%) is interested in a new point of care test. A delay time of 20 minutes is acceptable for 81% of the questioned persons. Other minimal criteria are sensitivity (90%), accuracy (90%) and the possibility to evaluate other new important markers. No clear conclusion could be given about the acceptable price of the equipment or device. In addition to this market research real patient samples of patients diagnosed with prostate cancer were collected. Total PSA concentration of these samples was centrally analysed by using the FDA approved Hybritech TandemR method. Following this central analysis samples were delivered to partners to test the samples by using both the commercial Biacore SPR-system and the new prototypes AW and iSPR instruments. The first results appeared to be promising but the time was too short to carry out a full clinical evaluation analysis of the new method. Novel SAW/SPR combined biosensor platform As an exciting new direction in acoustic wave based sensing, we have developed a novel biosensor system that integrates two different sensing techniques: Love mode Surface Acoustic Wave (SAW) devices and Surface Plasmon Resonance (SPR) [Friedt et al., Friedt patent]. The combined platform provides additional information related to the thickness, the water content and the adsorption kinetics of protein layers. This allows for the determination of the physical properties (i.e., density and optical index) of physisorbed layers of proteins onto a common sensing surface. The platform exploits the unique geometrical setup of the SAW device, which leaves the backside of the quartz wafer free of electrodes which allows to inject a laser in order to generate an evanescent surface plasmon on the gold coated sensing area. Such a configuration enables simultaneous estimation of various properties of the bound mass during electrochemical and biochemical reactions occurring on the common sensing electrode. These include the water content, film thickness and rigid verses viscoelastic characterization of the adsorbed films [Friedt patent]. In addition, determination of the water level content provides a more accurate measurement of the physical parameters of the protein layer itself (i.e. density and optical index) by compensating the influence of water once it has been identified [Friedt et al.]. REFERENCES: J.-M Friedt, L. Francis, G. Reekmans, R. De Palma, A. Campitelli and U.B. Sleytr. Simultaneous surface acoustic wave and surface plasmon resonance measurements: electrodeposition and biological interactions monitoring. Accepted for publication in Journal of Applied Physics, 2004. Friedt J.; Campitelli A.; Francis L.; Laureyn W.; Apparatus and methods for simultaneous surface acoustic wave and surface plasmon resonance measurements. Patent application. Nanostructured S-layer technology A well controlled, nanostructured biochemical interface, which provides optimum conditions for sensitive, reliable, and fast detection of antigens (PSA) was a basic concept of the PAMELA sensor. The envisaged design consisted of S-layer proteins as a crystalline linking layer at the transducer surface and PSA specific camel antibody fragments (VHH) bound to it in a controlled, highly organized manner with high stability and reproducible density. S-layer proteins represent a unique biological self-assembly system capable of re-crystallising at inorganic surfaces such as silicon, gold, or glass. A large number of functional domains in defined position and orientation enable S-layers to specifically interact with bio-molecules, thus providing a powerful and versatile molecular construction kit that can be optimised to a variety of different biological sensing applications. One significant challenge concerned the seamless integration of the biological layers in the MST fabrication process for realizing the overall uTAS modules. In order to achieve successful integration, careful system level design, incorporating all the different technological dimensions and disciplines, was critical. Mouse mAbs bio-engeneered against PSA As companies with R&D and marketing skills in the field of prostate cancer the role of Eurogenetics NV first and of DiaMed Eurogen NV later in the course project was to guide the consortium towards a biosensor concept for the screening for prostate cancer. This concept that was achieved at the end of the project had to be at the same time efficient in relation to the clinical aspects and cost effective in order to be competitive of the market. In order to achieve this goal a conventional ELISA system for the measurement of free and complexed PSA was used throughout the complete development. The company also generated new and better performing monoclonal antibodies specific for a series of epitopes on complexed and free PSA in such a way the that epitope array profiling of free and bound PSA in patients blood would be possible, as worked out in array sensor format by MESA. Besides providing an amplification method through colloidal gold conjugation, essential to the feasibility of the method DiaMed also engineered existing conventional monoclonal antibodies into recombinant single chain Fabs for anti PSA clone 3E6 in order to compare its performances with Camel Abs bio-engineered by the VIB partner. Finally DiaMed selected a pair of conventional monoclonal antibodies i.e. clones 1A7 and A67 which will allow to establish an ultra-sensitive biosensor device effective for screening of the PSA status in blood of an elderly male population at risk for prostate cancer. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
