The project is funded by the BMBF over the period 2013-2018.
Project Management: Prof. Dr. Peter Schierack and Dr. Stefan Rödiger
As part of the predecessor project InnoProfile "New technologies for molecular diagnostics - real-time PCR array", funded by the BMBF over the period 2007-2012, a device platform for applications in basic research and medical diagnostics was developed. This so-called "VideoScan" technology is based on fluorescence microscopy and is used for the automated and standardized detection of biomolecules, microparticles, bacteria, eukaryotic cells and other fluorescent objects.
The technical goals of the InnoProfile Transfer project "Image-based Assays" are to expand the application possibilities of VideoScan technology. These include the application of microparticle systems under controlled thermal fluctuations (e.g. PCR), the investigation of new cell-based detection systems (e.g. autoimmune diagnostics) and the combination of microparticle-based with cell-based detection methods.
An important aspect is the application and development of methods from statistics and bioinformatics. Within the scope of our work, we deal with the analysis of multivariate non-linear data sets. These occur, for example, in multiplex quantitative real-time PCRs, but also in melting curve analyses and the microparticle arrays we have developed.
Some of the questions to be answered are:
- How high can the multiplex degree of a microparticle-based real-time PCR be using different technical approaches?
- How fast, robust, reproducible and automated can microparticle assays be processed?
- How do I detect fast and cost-effective infectious pathogens via cell-based systems?
- How can I describe and combat bacterial biofilms?
- Can I use new fluorescent dyes, staining protocols and new standards to improve the validity and reproducibility of an assay?
- Does the coupling of cell-based assays and microparticle-based assays increase the diagnostic value of an assay?
The financial relief of the health care system is one of the most important challenges of the present. In diagnostics, intensive efforts are therefore being made to examine the samples simultaneously for several parameters in order to counteract the constant cost pressure and lack of time. The trend of the past years is clearly towards personalized medicine. In addition, sample material, such as cerebrospinal fluid, is rarely available in large quantities, so that as many tests as possible have to be carried out with as little sample material as possible in order to clarify a disease. This is referred to as multiparameter diagnostics.
Existing technologies enable the differentiation of certain virus types, but they are often difficult to process, have little potential for multiparametric analyses or cannot be used in a modular way.
At the Brandenburg Technical University Cottbus-Senftenberg, which was founded in July 2013, the InnoProfile Transfer Initiative "Image-based Assays" continues its successful work in the field of multiparameter diagnostics with a multidisciplinary team. The working group is headed by the foundation professor Dr. Peter Schierack and Dr. Stefan Rödiger. This was preceded by the five-year InnoProfile "New Technologies for Molecular Diagnostics" headed by Prof. Dr. Christian Schröder and Prof. Dr. Peter Schierack. Here, innovative detection systems for VideoScan technology were developed.
The VideoScan technology combines a variety of analytical methods into a fully automated fluorescence microscope system that works on the basis of digital image processing and is suitable for high throughput applications. This allows temperature-controlled cell and microparticle assays to be processed. Diagnostic questions are defined in so-called modules, so that DNA microparticle assays, protein microparticle assays (e.g. detection of autoantibodies), cell assays (e.g. algae, mammalian cells) and assays in solution (e.g. enzyme reactions) can be set up with the VideoScan technology.
A philosophy that was anchored early on is the focus on clinically relevant questions, which are closely coordinated with the long-standing industrial partners Attomol GmbH, Generic Assays GmbH, Medipan GmbH and PolyAn GmbH.
The InnoProfile Transfer project "Image-based Assays" provides an excellent research environment and paves the way for the transfer of results to a closed value chain. All work is carried out in close cooperation with the InnoProfile-Transfer-Initiative "Multiplex-Bead-Assays".
The project funding contributes significantly to the fact that multiparameter diagnostics is establishing itself more and more as a competence in the Lusatian region. Just a few years ago, the company Attomol GmbH and biotechnology at the former Lausitz University of Applied Sciences (FH Lausitz) were only known to experts. The initiative has made Senftenberg's developments in the field of multiparameter diagnostics nationally and internationally visible.
Together with industrial partners, the research results are to be transferred from proof-of-principle status into mature products for global marketing. All participants agree that value creation in the field of multiparameter diagnostics has only just begun. Last but not least, new jobs have been created and the training of sought-after specialists for clinics, research institutions and companies has been established.
Technical goals are the further development of microparticle systems for use under controlled temperature regimes (microparticle-based multiplex real-time PCR and melting curve analysis), the further development of cell-based detection systems and the combination of microparticle-based with cell-based detection methods for routine diagnostics, but also for the life science sector.
Microparticle-based real-time PCR technologies
In the previous project, a functional, automated measuring system was established, in which the unit of hardware and software enables real-time measurements under defined modulated temperatures. The principle was applied in multiplex real-time PCRs and melting curve analyses on microparticles.
Using microparticle-based real-time PCR, human papilloma viruses, point mutations in thrombophilia and infectious agents such as Escherichia coli are now to be detected and differentiated. The company is currently working on the next generation of hardware and software, which will lead to an increase in sample throughput and turn the next step into a marketable product.
On the hardware side, an expansion of the number of cavities is planned so that 32 cavities will be available for diagnostic applications. The degree of multiplexing of real-time PCRs is generally limited as the increasing complexity of reaction components leads to competing processes. Approaches are the reduction of complexity by creating defined microreaction spaces (emulsion PCR) or by coupling primers to microparticles (solid phase PCR). In addition, innovative probe systems such as the LoopTag PCR system are transferred to the microparticle assay. The LoopTag PCR system is a patented technology of the project partner Attomol GmbH. So far it was used for the application in solution. A transfer of this probe system to a microparticle-based platform offers the prospect of a significantly increased degree of multiplexing.
Innovative bacterial and cell assays
In medical diagnostics, bacteria are detected by cultivation on culture media and by detecting bacterial DNA. The InnoProfile Transfer project intends to develop fast and cost-effective methods using cell-based and microparticle-based systems in order to be able to describe complex biological systems.
It is important to be able to describe the properties of bacterial populations in a multiparametric way. For example, the adhesion of bacteria to surfaces and host cells is very important in microbiology. The formation of bacterial biofilm is also of clinical relevance, as it can pose a health risk. Bacterial biofilms are deposits of bacteria on surfaces such as the tubes of medical equipment or artificial joints. Due to their multi-layered and complex structure, it is difficult to combat them with antibiotics. Manual analysis is extremely time- and cost-intensive. Using fluorescence microscopy technology, a detection method is being developed with which the behaviour of bacteria and the cellular reaction of host cells can be investigated in vitro using fully automated, standardised high-throughput methods.
The detection methods will be based on established methods such as fluorescence in situ hybridization (FISH) and live/dead staining. The research focuses on Salmonella and Escherichia coli and their interaction with human and porcine cell lines. The ambition is to define the degree of pathogenic properties of a bacterium in the host. The combination of cell-based and microparticle-based assays has a high diagnostic potential. This includes the ability to qualitatively and quantitatively detect secreted soil substances and cytotoxins on microparticle surfaces in a time-resolved manner. The work will concentrate on time-resolved detection and quantification of biomolecules in a homogeneous format.
Within the framework of the InnoProfile initiative, Brandenburg's SMEs as well as national and international research institutions and universities are involved.
Attomol GmbH, Lipten, Germany
Generic Assays GmbH
Carl Thiem Clinic Cottbus gGmbH
Lower Lusatia Clinic GmbH, Senftenberg
Academy of Medical Sciences Moscow (Russia)
Belarusian Medical Academy of Post-Graduate Education (Minsk, Belarus)
Titertek-Berthold Detection Systems GmbH, Pforzheim, Germany
Federal Institute for Materials Research and Testing, Berlin
Dresden University of Technology
Fraunhofer Institute for Biomedical Engineering, Potsdam-Golm
Vaccine plant Dessau/Tornau GmbH
Institute for Cardiac Diagnostics and Therapy GmbH, Berlin
Otto-von-Guericke University, Magdeburg
University of Leipzig
University of Potsdam
University of Environmental and Life Sciences Wroclaw (Poland)
Laboratory physicians Sindelfingen
Brandenburg Technical University (BTU) Cottbus - Senftenberg
Faculty of Environment and Natural Sciences
Prof. Dr. habil. Peter Schierack, Stiftungsprofessor
Dr. Stefan Rödiger, Head of the Working Group "Image-based Assays"