Project: MIniaturized Detection Approach for pathogenic bacteria in fluids
Reliable and rapid methods for identifying the presence of pathogenic bacteria in food, water and organic fluids are crucial to safeguard human health. In the case of Salmonella, the conventional diagnostic serological methods are labor-intensive and require several days, a delay that impairs enforcing the remedies in due time for diseases such as acute infectious diarrhea and typhoid fever (1,2; References in the annex). New approaches based on a large variety of theoretical backgrounds are constantly proposed to reduce the time necessary to reach suitable diagnoses and to simplify the analyses(3-7). Most of them rely on immunoassays (i.e. ELISA) or molecular methods such as real-time PCR step that amplifies the coding regions of marker proteins (1,8-12). However, even PCR methods requires a preliminary enrichment phase in which the bacteria are overgrown(13) to achieve detection. The drawback of such enrichment phase is that it is apparently selective, since different strains showed remarkable growth variability when cultured in the standard media (14). Furthermore, some broth components can have inhibitory effects on the PCR itself (15). As an alternative, the enrichment of the templates has been successfully obtained by introducing an affinity purification step before quantitative PCR (16,17). A further improvement considers enriching the bacteria sample by immobilization on a gold surface using tailspike proteins for the capture. The binding effectiveness was monitored by surface plasmon resonance and strain specificity probed by PCR (18). The large number of proposed protocols indirectly indicates that no golden standard has been reached so far and that a robust and cost effective detection platform is still to be established. _x000D_In order to develop an optimized detection system for pathogenic bacteria the following hypothesis were considered: a) bacteria can be enriched by immunocapture, also on activated surfaces, avoiding selective pre-culture in ad hoc media(16,18); b) bacterial membrane proteins are apparently very suitable markers for detection (11,12); c) microfluidic chip technology can increase sensitivity and reduce the costs, while allowing automation for screening at large scale (19); d) different fluorescent probes can be used to identify specific bacterial strains in a mixed enriched population (4)._x000D_We propose the realization of a miniaturized diagnostic platform for the capture and detection of the pathogens and Salmonella will be used as a model organism. The system consists of a microfluidic device composed by a polymeric pad and a slide with a functional coating of nanostructured (ns-MeOx) metal oxide (20, 21) on which Bacteria Exposing Recombinant Antibodies (BERA) are immobilized. In this configuration, all the available antibodies are oriented outwards and, therefore, optimally available for interacting with their specific target antigen. The antibody preparation is straight forward since no purification steps are necessary: BERA are simply recovered from the culture medium, washed, and immobilized on the ns-MeOx surface. The reliability of such technology has been recently demonstrated for the selective binding of target proteins present in a complex solution (purification independent microarray-PIM technology, 22), whereas the identification of recombinant antibodies specific for pathogens has been repeatedly reported(23). The expression of recombinant antibodies raised against membrane markers would allow the capture of bacteria exposing those epitopes. Contacting the sample over the ns-MeOx-BERA surface will lead to a selective binding and enrichment of the target microbial cells. A labeled antibody, specific for a second and independent epitope, will be loaded successively for detecting the captured pathogens. The presence of largely expressed detection epitopes on the captured cells will amplify the signal, decreasing or eventually eliminating the conventional enrichment growth step and therefore reducing assay duration. A robotic liquid handler with customized solutions will be employed for loading the samples as well as performing the successive steps minimizing the processing time and reagent costs while providing automation . The device is flexible in terms of target specificity (bacteria of different species, viruses, or aptamers contaminating a fluid or recovered from solids in a fluid), simple to use guarantees reproducibility and will be designed to allow large scale screening for pathogen detection._x000D_Ps of this proposal already possess the theoretical and technological background necessary to develop the project into a mature product, namely the competence in producing immobilization surfaces using industrial quality standards (Tethis)(20,21), a one-pot library for specific antibody recovery (UNG)(24,25), the establishment of microfluidic robotic and methods for immunodetection (Tethis), and the managing expertise to bring ideas into the market (Tethis). _x000D__x000D__x000D_
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Acronym
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MIDA
(Reference Number: 6218)
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Duration
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16/04/2011 - 30/06/2015
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Project Topic
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The proposal plans to develop a miniaturized chip-based approach for efficient diagnostic detection of pathogenic bacteria and contaminants in fluid and solid samples by immunoaffinity. The method is designed to obtain a cost saving tool suitable for automation and large scale screening.
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Network
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Eurostars
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Call
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Eurostars Cut-Off 5
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Project partner
