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An integrated microbiosensor system for patient monitoring by measurement of extremely low analyte concentrations present in the blood

Objective

The aim is a more efficient system for monitoring patients diagnosed with prostate cancer, by measurement of the Prostate Specific Antigen (PSA) level in the blood. It is foreseen that the new technology will have an overwhelming impact in related areas of immunodiagnostic testing. Demand is a new, fully automated instrument to deliver in real-time, reproducible values for nanogram PSA levels per ml. blood.

A sensitive, miniaturised and integrated biosensor-system (TAS), for bioaffinity systems is made by a combined effort of optimisation of all elements and interfaces:
1) transducer,L%2) transducer/biological interface and biological probe molecules, and
3) microfluids.

Global integration results in the new analytical instrument based on the TAS incorporating either a novel acoustic wave (AW) system or the established surface plasmon resonance (SPR) technique. In the development, considerations regarding cost, lifetime and commercialisation potential are seen as a priority.

Objectives:
An improved method for the determination of PSA as set forth in the proposal will display the following characteristics:
1) Analytical sensitivity: nanogram per millilitre
2) Response time: 20 minutes (Real-time)
3) Analytical sample: drop of plasma or serum
4) High automated instrument for reproducible results.

The challenge of this project is to develop the required optimisation and combine them into an integrated device that can meet the clinical specifications as required by the market. The system will be of generic nature and can be easily adapted to other clinical analyses and environmental monitoring. The consortium, as well as the individual partners, intend to commercially exploit every technical improvement that will be identified during the course of the project in the various different research areas, e.g. in the biosensor, clinical analysis and lab-on-chip areas.

Work description:
The new TAS will integrate diverse components.
1. Stable and high affinity PSA-specific biomolecular probes derived from camel antibodies. The antibody-derived molecules will be engineered to allow selective chemical manipulation and hence oriented immobilisation at the sensor surface.
2. An optimised transducer/biology interface improving signal to noise level and hence sensitivity. An efficient modification of the transducer surface will be achieved by re-crystallisation of so-called S-layer proteins that generate a regular pattern of chemically reactive groups at the surface. S-layer technology has not been applied to industrial processes, yet. Therefore, attention will be paid to the adaptability of the crystallization procedure for industrial scale and the compatibility with subsequent fabrication steps. To identify critical process parameters and to study the quality of S-layer deposition and antibody immobilization on S-layer modified surfaces, detailed state-of-the-art in situ and ex-situ surface analysis will be performed.
3. An AW sensor of high sensitivity. Development of extremely sensitive AW sensor modules that will "plug" into the overall instrument. Modules have sufficient package stability for manual handling by non-technical users.
4. Microfluidics allowing direct injection of serum or plasma sample and with concentrating effect on the analyte.

The overall instrument performs the automatic control of the TAS, i.e. the analyte sampling and the sensor data acquisition. To improve the progress of the investigations, the value of the different optimisations is tested by implementation of a SPR system. A comparison in the effectivity between the adapted SPR and the new AW system, in terms of clinical performance and compatibility to industrial production, will facilitate the selection of the instrument that should be further commercially exploited.
Significant scientific, developmental and commercial achievements were realised during the course of the PAMELA project, even though the consortium faced many technical challenges during the critical integration phase of the project. Of note, the development of the sensor modules based on the SAW and SPR transducers proved more challenging than originally expected at the start of the project. However, the resulting technology path undertaken led to highly original solutions with the initial prototypes providing some very promising results. The PAMELA concept has evolved significantly over the duration of the project. The original concept back in 1999 for the PAMELA instrument was to be capable of providing an informed "YES/NO" decision concerning the presence of the PSA (at a concentration threshold of 4ng/ml). The sensor modules were to be based on the mTAS concept, integrating the microfluidics, surface chemistry and the transducers (either SAW or SPR). The biochemical interface was based on S-layers with immobilised selective receptors from camel antibodies engineered specifically against the target PSA molecule. This rather simple concept proved more challenging that originally anticipated, and by the end of project (31 March 2003), the technological direction had changed dramatically.

Both the SAW and SPR transducers had evolved to more sophisticated designs, whereby novel configurations were implemented. For the SAW device, this lead to the development of a new biosensor platform that combined both the SAW and the SPR sensors on the one common sensing surface. This highly innovative configuration allows for multi-parameter analysis of the sensing event, providing additional information related to the thickness, the water content and the adsorption kinetics of protein layers. For the SPR configuration, a new imagining concept was introduced that allows for an array of SPR data, hence facilitating a "fingerprint analysis" of the sensing event. As a direct result of this novel technique, partner HBM developed, marketed and launched a new product called the iSPR instrument. The biological interface also dramatically improved from the original concept. The development of the S-layer technology provided a very attractive route for the creation of the sensitive interface. Two strategies towards an ordered bio-specific interface were developed: the covalent immobilization of PSA specific cAb fragments on S-layer (SbpA) coated transducer surfaces; and coating the transducer with a novel fusion protein between an S-layer protein and a PSA specific cAb fragment. Both techniques addressed the important issues of sensitivity (towards the target PSA) and selectivity (against non-specific adsorption). Mixed SAMs of thiols were also investigated for their enormous potential as building blocks for the implementation of highly effective biochemical interfaces. The realised surfaces proved to be highly ordered, well controlled and reproducible, leading to enhanced sensor performance in terms of the sensitivity and selectivity. For the selective receptors themselves, specially engineered camel antibodies against PSA (total) and mouse monoclonal antibodies against the different PSA epitopes were realised. This offers great flexibility and enhanced array sensing capability for the development of a true "fingerprint analysis" of the PSA sensing event. An overview of the main technological achievements of PAMELA is summarised as follows: *Camel Abs bio-engineered against PSA (total) *Mouse mAbs bio-engineered against PSA (epitopes) *Nanostructured S-layer technology *Analytical charaterisations bio-interface *iSPR instrument developed + commercialised !! *SAW sensors developed *PSA sensor response 1 ng/ml (SPR), 100 ng/ml (AW) *SAW/SPR platform validated with patient serum samples As an outcome of the PAMELA project, the potential "patents and products" are summarised as follows: PATENT APPLICATIONS: *Molecular glue (LISE) *iSPR (HBM + MESA+) *S-layers (ZUF) *cAbs (VIB) *Fluidics (MESA+) *SAW/SPR platform (IMEC) PRODUCTS: *iSPR (HBM + MESA+) *mAbs (DiaMed Eurogen) *cAbs (VIB) *S-layers (ZUF) A summary of the valuable technical and management lessons learnt from the PAMELA experience can be summarised as follows: Technical lessons *Integration, integration, integration !! *Flexible, adaptable engineering solutions *Understanding of interfaces Management lessons *Large consortiums = huge opportunities, but challenging *Co-ordination separate SCIENCE + ADMINISTRATION *Consortium Agreement signed + sealed ASAP before start *Clear common consortium vision at all times Overall, PAMELA has been a highly successful and visionary project with significant outcomes. Of note, it was used as the leading example in the recently published NEXUS Association Roadmap for Medical devices as a MNT applications for Point-of-Care diagnostics.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

INTERUNIVERSITAIR MICRO-ELECTRONICA CENTRUM VZW
Address
Kapeldreef 75
3001 Leuven
Belgium

Participants (9)

AVENTIS PHARMA B.V.
Netherlands
Address
Bijenvlucht 30
3871 JJ Hoevelaken
BUNDESANSTALT FUER MATERIALFORSCHUNG UND -PRUEFUNG
Germany
Address
Unter Den Eichen 87
12205 Berlin
DIAMED EUROGEN
Belgium
Address
Hertoginstraat 82
2300 Turnhout
FACULTES UNIVERSITAIRES NOTRE-DAME DE LA PAIX
Belgium
Address
Rue De Bruxelles 61
5000 Namur
HOLLAND BIOMATERIALS GROUP BV
Netherlands
Address
Drienerlolaan 5
7522 NB Enschede
NANOSEARCH MEMBRANE GES.M.B.H
Austria
Address
Hettenkofergasse 13
1160 Wien
UNIVERSITAET FUER BODENKULTUR WIEN - ZENTRUM FUER ULTRASTRUKTURFORSCHUNG
Austria
Address
Gregor Mendel Strasse 33
1180 Wien
UNIVERSITEIT TWENTE
Netherlands
Address
Drienerlolaan 5
7522 NB Enschede
VLAAMS INTERUNIVERSITAIR INSTITUUT VOOR BIOTECHNOLOGIE VZW
Belgium
Address
Rijvisschestraat 120
9052 Zwijnaarde