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FP6

SLIC Informe resumido

Project ID: 513771
Financiado con arreglo a: FP6-LIFESCIHEALTH
País: Switzerland

Final Report Summary - SLIC (SLIC-biosensors in molecular diagnostics: nanotechnology for the analysis of species-specific microbial transcripts)

The key aim of the SLIC project was the development of a novel biosensor-based device for molecular diagnostics for application in clinical microbiology. Current state-of-the-art technologies for molecular diagnostics have technical limitations, are expensive and sometimes require highly skilled personnel and heavy infrastructure. It was a primary aim of the project to focus on post genomic applications, i.e. non-deoxyribonucleic acid (DNA) applications, which in this case was transfer-messenger ribonucleic acid (tmRNA). The simplification steps envisaged in the project would make ribonucleic acid (RNA)-based tests faster, easier to use, cheaper and thereby making them more globally accessible to both developing and developed healthcare markets.

Towards achieving this objective, the SLIC consortium set out to accomplish the following key tasks:
- Sample preparation - integration of cell lysis and RNA purification/extraction in one step. Normally these are several discrete steps that can be time consuming and are very prone to contamination and RNA material loss, thus making them very critical for the ultimate RNA yield and overall assay accuracy.
- High-performance capture probe design - the accuracy of any diagnostic assay rests considerably on the performance of the capture probe. Furthermore, probe hybridisation typically requires certain stringent conditions, e.g. higher temperature, for it to work optimally, which adds to overall assay complexity and cost. The capability to design better probes that could even function close to body temperatures was another key objective of the project.
- Ultrasensitive detection - typically, assays based on nucleic acid targets require amplification steps, in order to render them detectable with standard detection technologies. However, this adds appreciable complexity and cost to the overall assay, hence the project set out to substitute this with a novel ultrasensitive biosensor approach based on the SLIC technology.

The development of the novel biosensor-based device for application in molecular diagnostics was based on combining two proprietary technologies, the SLIC-Nanobiosystem, the biosensor platform and Riboseq, a molecular target technology. The SLIC-Nanobiosystem consists of a self-assembled lipid bilayer membrane that integrates a synthetic ligand-gated ion channel (SLIC). The SLIC comprises a capture molecule that can specifically bind a given analyte, a process that is monitored via electrical impedance spectroscopy.

The Riboseq platform is based on the universal bacterial genomic target, tmRNA, which is encoded by the ssrA gene. It is a high copy number RNA target that has conserved and variable sequence signatures, which can be exploited to develop nucleic acid tests for microbial identification. S. pneumoniae was selected as the initial model system for the development of the biosensor-based device.

Since the capture probe also plays a key role in overall assay performance, a special effort was given to develop new tools for optimising tmRNA capture probes. These tools would also be used to develop probes that could simplify assay protocol, e.g. provide probes that can hybridise at lower temperatures (e.g. body or room temperatures).

Sample preparation is central to any diagnostic assay, but remains one of the areas that have been poorly addressed so far for RNA-based diagnostics. Therefore, significant effort was accorded for developing a novel platform that could carry out cell lysis, RNA purification in a rapid automated step. It was envisaged that such a novel sample preparation device would be integrated with the SLIC biosensor downstream, in order to provide a homogenous assay format.

The tasks towards these objectives were achieved through the various work packages (WPs) outlined below:
- design of DNA probes for pathogen detection (WP1);
- development and characterisation of SLIC detection platform (WP2);
- optimisation of nucleic acid detection in the biosensor (WP3);
- development of a sample preparation device for nucleic acid extraction (WP4);
- performance evaluation of the biosensor-based device for clinical testing (WP5).

The consortium successfully developed panels of DNA probes based on the Riboseq technology for the model pathogen S. pneumoniae and other target pathogens, H. Influenza and M. tuberculosis. Specific DNA probes for S. pneumoniae were identified, selected and applied to develop and demonstrate various nucleic acid based diagnostic formats including biosensor, microarray and real-time in vitro amplification based (NASBA) formats. The application of Riboseq as a molecular target technology for microorganism identification was thus successfully demonstrated. Nucleic acid test formats including direct hybridisation dual probe sandwich assay were shown to be compatible with biosensor based detection, although detection of nucleic acids in this platform was not necessarily at clinically relevant levels.

The demonstration of the Riboseq technology as a diagnostic target is an important achievement. Due to its occurrence in high copy numbers, it represents an appreciable step towards reducing the burden/requirement of intermediate target amplification steps, especially as biosensor technologies continue to evolve and improve. In combination with the SLIC capture probe optimisation tools (e.g. SLICsel), it promises to provide a powerful technology base for improved molecular diagnostics tests for various key infectious diseases, such as respiratory diseases, and may also find relevance in bioterrorism diagnostics.

The SLIC RNA extraction platform represents a new breakthrough in the area of enabling tools for RNA analysis, especially small RNAs, one of the most intensely researched emerging domains. It is expected to attract interest primarily in drug discovery and biodiagnostics. In drug discovery, it provides equal or better small RNA recovery efficiency, but at a fraction of the time, than the leading products (e.g. Ambion) in the market does. In point-of-care testing (POCT), according to a recent review of emerging technologies for nucleic-acid POCT, currently all the instruments in the market focus on DNA detection.

Therefore, the SLIC RNA platform may have an opportunity to be one of the first RNA POCT devices in the market. A patent has been filed and will form the basis of a commercialisation plan that was elaborated by the SME partner in the consortium.

Información relacionada

Contacto

Solomzi MAKOHLISO, (Chief Executive Officer)
Tel.: +41-216938631
Fax: +41-216938631
Correo electrónico
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