Diagnostic potential of tiny vesicles

Within the last years the interest in extracellular vesicles (EVs) increased dramatically due to their capability to represent their cell of origin during the moment of release. Within our group we investigate this feature as a promising approach for diagnostics in liquid biopsy. Although EVs are only 30 nm up to a few micrometer in size they are enriched in multiple molecules (proteins, nucleic acids, lipids, metabolites, among others). Those potential biomarkers are surrounded by a lipid bilayer protecting them against extracellular degradation. In addition, EVs are continuously secreted from virtually all cells and are consequently found in all bodily fluids (e.g. blood plasma, urine, saliva). Thus, non- to minimally-invasive isolation is possible and conventional tissue biopsy could be avoided in the future.

At the Fraunhofer IZI an antibody microarray was established to analyze the expression of EV surface markers in malignant diseases from different bodily fluids. The findings are valuable for the enrichment of tissue- or disease specific EVs. The aim is to separate the relatively small fraction of disease-associated EVs from the broad background of "healthy" EVs and to study their cargo. A correlation within the EVs and the tumour cells is expected, however, additional changes in the profile might be identified which could improve or perhaps even render diagnostic or therapeutic decision making possible in the future.

Characterization and testing of multisensors using cell engineering methods

The aim of the project is to research molecular acoustics applications for the quantitative and qualitative classification of blood, blood plasma and dialysis filtrates. The project will also look at how emitted acoustic waves propagate in bodily fluids. Specific characteristics will be identified that pinpoint the tiniest differences in the composition of the investigated medium. Moreover, it will also be characterized to what extent multisensor systems used in vivo are able to carry out non-destructive real-time analyses and therefore surpass existing diagnostic procedures in terms of speed and precision. Besides acoustic sensors, optical and magnetic sensors are also to be used.

logo EFRE Saxony
Saxon State Signet

This project is co-financed by tax revenues on the basis of the budget approved by members of the Saxon state parliament.

Diagnostics of various diseases in exhaled air using ion mobility spectrometry

IMS silicon chip of the Fraunhofer IPMS
© Fraunhofer IPMS
IMS silicon chip of the Fraunhofer IPMS.

The diagnosis of diseases should be as quick, easy and inexpensive as possible, directly at the point of care without highly qualified laboratory personnel and also without additional stress for the patient. To meet the existing requirements, research is being carried out at the Fraunhofer IZI and the Fraunhofer Project Center MEOS on a diagnostic method using exhaled air.

Breathing gas analysis has been used clinically for years as part of lung function tests or the urea breath test to detect Helicobacter pylori infections. However, there is a much greater potential in breathing gas diagnostics. There are thousands of so-called volatile organic compounds (VOCs) whose composition in the air we breathe changes with certain diseases and infections. For example, the diabetic patients’ smell of overripe fruit is known, caused by a greatly increased concentration of acetone, a VOC.

Most VOCs occur in extremely low concentrations (ppmV to pptV - one particle per million or trillion particles). This places extreme demands on the measurement technology. Conventionally, mass spectroscopic methods are mostly used, which, however, mean an enormously high expenditure on equipment. We therefore focus on ion mobility spectrometry (IMS), which is coupled with gas chromatographic (GC) pre-separation. This technology is much cheaper, more portable (shoebox size) and is already being used for drug and explosives controls.

In Leipzig, GC-IMS was able to differentiate between different bacteria based on their VOC profile after only 90 minutes of cultivation in the laboratory in 2019. In the course of the project, these experiences will now be used to directly examine real breath samples in the clinic. The aim is to diagnose various bacterial and viral infectious diseases, including any existing drug resistance, in patients’ breath.

In the MEOS project center in Erfurt, colleagues from Fraunhofer IZI and Fraunhofer IPMS are working on a new IMS system. The centerpiece is a small IMS silicon chip, which is being further developed at Fraunhofer IPMS in Dresden. Potentially miniaturized IMS chips can be inexpensively manufactured in large quantities using the microelectronic manufacturing processes. The new system is to be tested in the diagnosis of neurological diseases and cancer. In addition to the exhaled air, other non-invasive samples such as sweat and urine are also in focus.

Explanation of the IMS measurement procedure which is being developed at the Fraunhofer Project Hub MEOS. Video realization © 3D Agentur Berlin, Dirk Puder und Stefan Loth, GbR

Non-invasive diagnostics by breath analysis

Subject test for breath analysis
© Fraunhofer IZI
Subject test for breath analysis.

Exhaled air contains substances known as volatile organic compounds (VOCs), which provide information about metabolism. In a variety of diseases, including infections, cancer and neurodegenerative diseases, the metabolism and thus the composition of the exhaled VOCs changes. Detection of these VOCs offers the opportunity to diagnose diseases early and non-invasively.

Ion mobility spectrometry (IMS) can detect VOCs within minutes directly at the point-of-care. The BMBF project ”Breath Alert” investigates whether IMS can be used to detect antibiotic resistance in bacteria. In the Fraunhofer-versus-Corona cluster project ”M3Infekt”, IMS technology was further developed at the Fraunhofer Center for Microelectronic and Optical Systems for Biomedicine (MEOS) with the participation of Fraunhofer IZI. Specifically, methods for sampling via mouth and nose, for short-term sample preservation and for sample preparation were established and tested. At the end of the project, the method was tested on 60 healthy volunteers in two clinical studies in Dresden and Magdeburg. In parallel, a functional novel IMS demonstrator was completed at MEOS. This must now be further developed and optimized in follow-up projects to selectively detect diagnostics-relevant VOCs in complex matrices such as exhaled breath.

In the M3Infekt project, the participating nine Fraunhofer Institutes developed further non-invasive and mobile sensors for recording heart rate, ECG, oxygen saturation, respiratory rate and respiratory volume. Concepts for system integration and flexible interfaces were defined and a multimodal AI framework for cross-sensor data evaluation was developed. In addition, requirements regarding conformity to medical regulatory requirements were developed. The overall vision of the project is a close monitoring of relevant clinical parameters for detecting condition deterioration in infectious diseases also outside of intensive care units via a multimodal, modular and mobile sensor system. During the project it has become apparent that several specific solutions for different sub-applications are more useful than a single overall system and thus the benefit of the project results is even increased.

BreathAlert – Detection of antibiotic-resistant bacteria using ion mobility spectrometry

In a report, the World Health Organization emphatically describes why the fight against antibiotic resistance is one of the major tasks faced by the global community. By the year 2050, the organization expects 10 million deaths annually that will be attributable to infectious agents [1]. Innovations are necessary not only for therapeutic treatment, but also for the diagnostic detection of pathogens that cause disease to counteract the challenge in the healthcare sector.

The BreathAlert project launched at the end of 2020 aims at improving this situation with a new method for the rapid and non-invasive detection of infectious agents and antibiotic resistance, which analytically analyzes patients’ breath. The project focuses on the further development of ion mobility spectrometry, which is to be used to characterize volatile organic components (VOCs) of microorganisms.

At the Fraunhofer IZI, selected microorganisms are being examined to determine whether they can be differentiated via the VOCs released and whether they can be assigned to the respective types of bacteria. For this purpose, the pathogens are first cultivated and then the headspace, the gas phase above the culture medium, is fed into the device. The VOCs are ionized, separated in an electric field and then detected at different times. Software analyzes the complex data. The aim is to identify specific signals with which bacteria can be reliably differentiated from one another, even under different conditions. The focus is on antibiotic-resistant pathogens, such as enterobacteria, which are increasingly showing resistance to carbapenem and cephalosporin [1].

The characterization of clinical isolates, air samples from infected patients and measurements of the influence of e.g. eating habits on the air we breathe round off the project.

The company Graupner medical solutions GmbH, which develops the medical device technology, works together with the Fraunhofer IZI as a consortium partner. The development work is supported by specialist clinics that provide access to samples and also carry out a final validation. The project results are used economically by the Graupner company.

1] World Health Organization Report 2017: Prioritization of Pathogens to guide discovery, research and development of new antibiotics for drug-resistant bacterial infections, including tuberculosis. WHO/EMP/IAU/2017.12

HyFly – Non-invasive diagnostics for infectious diseases

Modern air traffic is not just useful for the fast transport of passengers or goods. Even infectious agents travel by planes over long distances within a few hours as unwanted passengers. Infectious diseases such as influenza or SARS, which can develop into pandemics, are nowadays spreading rapidly and much faster than years ago. Steps to effectively control and prevent chains of infection in the field of modern mobility have not yet been effectively established worldwide. Initial approaches using simple questionnaires or non-contact temperature measurement, however, have remained largely ineffective. The project "HyFly", funded by the German Federal Ministry of Education and Research (BMBF) within the InfectControl 2020 initiative, addresses the sensitive issue of passenger control. Together with partners, a non-invasive method based on ion mobility spectrometry (IMS) is being worked on. Applying this strategy, infected persons should be identified within a few minutes via components of their breathing air. IMS is already routinely used worldwide to detect drugs or explosive remnants in airports providing an established infrastructure. So-called volatile organic substances that are metabolic products of microorganisms are detected. The focus is on bacterial pathogens, which, according to information from the German Robert Koch Institute, have a high relevance for aviation. Initial results show that the method has great potential for discriminating between different pathogens. In addition to system development, a study is underway to identify microorganisms at various international airports to determine the cleaning efficiency and impact of antimicrobial coatings.

Sample Preparation

Sample preparation
© Fraunhofer IZI
Sample preparation

Sample preparation is a crucial aspect in many areas of bioanalytical research, especially in the analysis of the crude complex samples and/or rare targets. Modern laboratories exploit very sensitive methods of detection including molecular diagnostics; however, their performance strongly depends on the quality of the sample. Pre-analytical processing must prepare the specimen for the most effective detection of the target. It includes the purification of the analyte, its pre-concentration, as well as the parallel removal of the compounds which may affect the analysis. The preservation of samples which prevents the degradation of the target is also a task for this field of applied analytics. The main aim of sample preparation is to ensure the precision of the subsequent analysis. None of the sample preparation approaches are universal: they must take into account methods and equipment for the downstream processing of the specimen, concentration and nature of the analyte, volume of the sample and many other factors.

Nowadays bioanalytical research actively pursues integrating complex assays on automated platforms including lab-on-chip devices. This trend often lacks intelligent solutions for the pre-analytical steps. The aim of our working group is to support this particular field by developing of the most suitable sample preparation approaches for specific needs.

The group supports researchers and industrial partners with customized solutions, evaluates capacities of existing methods and develops novel strategies for effective pre-analytical processing.

Parodontitis chip

Taking samples for the rapid analysis of periodontalpathogenic agents using the parodontitis chip
© Fraunhofer IZI
Taking samples for the rapid analysis of periodontalpathogenic agents using the parodontitis chip.

Periodontitis is an inflammatory disease of the gums that, if left untreated, can lead to tooth loss. In Germany alone it is predicted that nearly 12 million people are affected by periodontitis. The main trigger for periodontal disease is bacterial plaque which can lead to a reduction of the dental bone tissue. The postulated systematic relationship between periodontal disease caused by bacterial pathogens and cardiovascular damage has been studied extensively. It can result in particularly serious diseases such as heart attacks and strokes.

The parodontitis chip project is aimed at developing a fully integrated diagnostics platform both for the fast processing and the subsequent analysis of periodontal pathogens in complex samples. This innovative technology consists of a compact microfluidic card and a combined purification module. Steps such as isolating pathogenic nucleic acids, selectively amplifying DNA sequences, and their specific detection are integrated to establish an easy-to-use setup for the end-user.

The lab-on-a-chip device will allow the detection and characterization of 11 bacteria relevant to the pathogenesis of periodontitis in a parallel format. In addition, the establishment of a simple detection unit will allow the monitoring of reaction kinetics. Therefore a quantification of the pathogen, as well as a determination of the total bacterial count can be realized.

The parodontitis chip project will allow for the creation of a simple molecular diagnostic test platform that can easily be adapted to various problems in the field of medical, environmental, or food analysis. Simplified lab-on-chip devices having an extremely simple structure and non-contact detection units provide significant time and cost savings for the user.

Development of diagnostic test strip systems

The significance of easy-to-use test strip systems for the rapid detection of clinically relevant parameters or for quality assurance of food products is increasing not only in developing countries. We develop a simple diagnostic platform that is particularly suitable for nucleic acid-based formats. As a reference assay, pathogens are diagnosed in human samples.

TBSearch project

Tuberculosis is an infectious disease, which is caused by Mycobacterium tuberculosis. According to a 2013 report of WHO (World Health Organisation), Tuberculosis ranks as the second leading cause of death from an infectious disease worldwide, after the human immunodeficiency virus (HIV).

An early and reliable diagnosis is crucial. Our unit is currently developing in cooperation with the McMaster University in Hamilton (Canada) a detection system which is rapid, simple, and cost-efficient. The nucleic acid-based test system integrates all steps from pathogen isolation to hybridization of nucleic acid on pathogen-specific probes.

Rapid on-site diagnosis of infectious diseases based on a lab-on-a-chip system

Micro fluidic card as CAD (Computer Aided Design) model
© Fraunhofer IZI
Micro fluidic card as CAD (Computer Aided Design) model.
Controlling magnetic nanoparticles through a microfluidic chip – produced by the rapid prototyping method – with the aid of magnets that are arranged above and below the microfluidic chip.
© Fraunhofer IZI
Controlling magnetic nanoparticles through a microfluidic chip – produced by the rapid prototyping method – with the aid of magnets that are arranged above and below the microfluidic chip.

A reliable diagnosis of complex and life-threatening infectious diseases (e.g. sepsis) is currently only possible using elaborate and time-consuming methods involving an analysis laboratory and qualified specialists. The Unit is developing an innovative system for rapid, easy-to-conduct and inexpensive on-site infection diagnostics.

The system is based on magnetic particles in the micrometer scale that can be functionalized according to their respective application to act as carriers for antibodies and disease-associated DNA sequences. These magnetic particles are employed on a disposable object in the approximate shape of a check card. In an on-site examination a sample is obtained from the patient, such as blood, saliva or urine, which is then incorporated into the lab-on-a-chip system. After lysis of the target cells, the magnetic particles bind to the respective target molecules in the sample and are transported via magnetic forces through different reaction tubes in a fully automated manner. At the end of the process chain the diagnosis is performed using a highly sensitive magnet sensor system.

The project is funded by the Federal Ministry for Education and Research (BMBF, Bundesministerium für Bildung und Forschung) and coordinated by Magna Diagnostics GmbH, a spin-off company of the Fraunhofer IZI.

NoDope – detection of doping misuse by athletes

Athletes have been trying to improve their performance through illegal means for decades. They use specific products that can enhance endurance, muscle growth and strength or improve recovery after extensive trainings/contests.

Within this project, a diagnostic device is developed to detect different doping products in blood samples. A complete integration of sample preparation and bioassay will be designed and developed. Furthermore, the power of this device will be the detection of multiple doping products in one assay based on surface plasmon resonance (SPR) technology.

Reference project: System validation for ApoStream™ technology

Circulating tumor cells (CTCs) are cancer cells shed from a primary tumor and circulated in the peripheral blood. Detection and isolation of CTCs can be used for diagnostics purposes and e.g. personalized medicines. Circulation tumor cells (CTCs) differ from peripheral blood mononuclear cells (PBMCs) in many ways. Detecting CTCs in blood samples is however a challenge, since CTCs comprise a very rare population among red blood cells and leukocytes.

The ApoStream™ technology from ApoCell can detect and enrich CTCs from blood with the use of a method called dielectrophoresis (DEP). This device is still in development and in order to validate the system, surrogates for CTCs and PBMCs need to be developed. Both biological as non-biological surrogates are designed, produced, screened and optimized.

Reference project: Luciferase-based reporter gene assays for functional genomics and drug discovery

Reporter gene assays offer a wide range of applicabilities in modern research. In the first place, they serve for characterizing regulatory elements (promoter regions) or modulators (transcription factors) on the genomic level. In particular, luciferase-based reporter gene assays are established in current biomedical and pharmacological research. Due to their extremely low detection limit they have prevailed over fluorescence-based reporter genes. Basically, the regulatory genetic element to be investigated is cloned upstream of a luciferase gene. The reporter gene construct can be introduced into a selected cell system by means of conventional transfection methods. The biological activity of the cloned genetic element can now be characterized in a time-dependent manner. The Fraunhofer IZI offers a complete dual-luciferase system that is suitable for the investigation of genetic regulatory elements from mammalian genomes.


The method comprises three steps:

  • cloning and sequencing the target sequence into the reporter construct
  • transfection of the selected cell system with the reporter construct
  • measurement of the biological activity

optional steps:

  • delivery of external stimulants (e.g. agents) or alteration of culture conditions (e.g. hypoxia)
  • generation of deletion mutants

Reference project

In a cooperation project with the department of General Biochemistry of the University of Leipzig we succeeded in investigating the activity of the human GLO1 promoter in different carcinoma cell lines. At present, the influence of hypoxia on promoter activity is being investigated.

Selected applications

The system is used for the functional characterization of any conceivable regulatory element from mammalian genomes. Moreover, the system offers a way of examining exogenous factors in combination with normal or altered culture conditions.

Reference project: RNA interference-based tumor models for substance screening and elucidation of mechanisms of action of potential agents

RNAi is a conserved mechanism that regulates gene expression on the post-transcriptional level. In eukaryotes, double-stranded RNA (dsRNA) is processed to form short, small interfering RNAs (siRNA) which results in the degradation of the complementary mRNA. Finally, this leads to the effective down-regulation of the specific protein. These characteristic features result in the application of RNAi in cell cultures and animal models. The specific suppression of gene expression and protein activity modulates the pharmacological inhibition of the target protein and is thus an effective tool in proof-of-principle experiments and the identification and validation of antitumor agents.

For providing a novel cancer therapy, the reference project aims at the specific and gradual down-regulation of an already identified target protein known as glyoxalase I (GLO I) by means of RNAi. Moreover, the system shall provide a way of re-up-regulating GLO I in order to recreate the initial phenotype.

In tumors there is a strong correlation between signal transduction pathways and fundamental metabolic pathways, such as glycolysis and the pentose phosphate cycle. Many tumor-promoting mechanisms have an immediate effect on glycolysis, on the cellular response to oxygen and on the ability of tumors to recruit new vessels for their own nourishment. Since Warburg it has been known that tumors consistently utilize anaerobic pathways to produce ATP by conversion of glucose. One cytotoxic byproduct of glycolysis is methylglyoxal (MGO). The reactive MGO binds to proteins and nucleic acids in high concentrations, thereby inducing apoptosis. All organisms have a glyoxalase enzyme system (GLO I / GLO II) to prevent cell damage caused by high MGO concentrations. In particular in tumor cells, this system is up-regulated in order to minimize the paracatalytically generated high MGO concentrations. The inhibition of glyoxalases could therefore play an important role in cancer therapy. To date, the characterization of the glyoxalases in malignant tumors only referred to histochemical analyses which show an overexpression in tumors. By means of the RNAi model, specific GLO I inhibitors could be identified in substance libraries.