Nanotechnology

This unit works on the development of molecular diagnostic test systems for the sectors food and medicine / clinical practice. In the first place, a novel lab-on-a-chip diagnostic platform is implemented on the basis of functionalized magnetic particles. An additional focus of this unit is on the development of test strip-based rapid tests, the characterization of nucleic acid-binding proteins and the evaluation of the toxic potential of industrial nanomaterials.

  • Magnetic bead-based assays
  • Lab-on-a-chip technology
  • BIAcore – Optical biosensors in molecular diagnostics
  • Nanotoxicology
  • Functional test strips
  • RNA interference
  • Reporter gene assays
  • Protein expression and purification

Sample Preparation

Sample preparation
© Photo 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.
© Photo 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.
© Photo Fraunhofer IZM

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.
© Photo 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.

Production and characterization of DNA-binding proteins

A plurality of different proteins exhibit specific DNA binding properties that are associated with targeted cellular functionalities. We utilize zinc finger proteins that bind specific nucleic acid sequences and are capable of capturing double-stranded DNA from sample solutions when used as immobilized components of a protein array.

Furthermore, we focus on functional proteins that are capable of isolating pathogens from an excess of human DNA. These proteins are intended to be employed in novel purification methods.

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.

Method

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.

  • AIT Austrian Institute of Technology GmbH, Department of Health & Environment, Nano Systems Business Unit
  • BECIT GmbH
  • DMCE GmbH & Co KG
  • ERT-Optik Dr. Thiel GmbH
  • Flensburg University of Applied Sciences
  • Fraunhofer Institute for Electronic Nano Systems ENAS
  • Fraunhofer Institute for Interfacial Engineering and Biotechnology IGB
  • Fraunhofer Institute for Reliability and Microintegration IZM
  • Institut für Mikrotechnik Mainz GmbH, Department Microfluidics
  • Leipzig University of Applied Science, Faculty of Electrical Engineering and Information Technology
  • McMaster University, Hamilton, Canada
  • Magna Diagnostics GmbH
  • Merck KGaA
  • microfluidic ChipShop GmbH
  • Research Center Borstel, Leibniz Center for Medicine and Biosciences
  • Seoul National University, NANO Systems Institute
  • Siemens AG, Corporate Technology, Electromagnetic Systems and Superconductivity
  • University of Leipzig, Faculty of Medicine, Institute for Biochemistry
  • University of Leipzig, Faculty of Medicine, Institute for Pathology

Publications

  • Gärtig C, Niemann K, Berthold J, Giel L, Leitschuh N, Boehm C, Roussak L, Vetter K, Kuhlmeier D. Development of a point-of-care-device for fast detection of periodontal pathogens. BMC Oral Health. 2015 Dec 24;15(1):165.
  • Tröger V, Niemann K, Gärtig C, Kuhlmeier D. Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems. Journal of Nanomedicine & Nanotechnology. 01/2015; 6(3). DOI dx.doi.org/10.4172/2157-7439.1000282
  • Sandetskaya N, Engelmann B, Brandenburg K, Kuhlmeier D. Application of immobilized synthetic anti-lipopolysaccharide peptides for the isolation and detection of bacteria. Eur J Clin Microbiol Infect Dis. 2015 May 19.
  • Sandetskaya N, Naumann A, Hennig K, Kuhlmeier D. Specific enrichment of prokaryotic DNA using a recombinant DNA-binding protein. Analytical and Bioanalytical Chemistry. 2014 Jun;406(15):3755-62. DOI dx.doi.org/10.1007/s00216-014-7787-7.
  • Sandetskaya N, Allelein S, Kuhlmeier D. Application of nanotechnology in miniaturized systems and its use for advanced analytics and diagnostics - an updated review. Recent Pat Food Nutr Agric. 2013 Dec;5(3):220-38.
  • Becker H, Carstens C, Kuhlmeier D, Sandetskaya N, Schröter N, Zilch C, Gärtner C. Stationary microfluidics: molecular diagnostic assays by moving magnetic beads through non-moving liquids. 20 Mar. 2013; SPIE Digital Library.
  • Sandetskaya N, Lorenzen M, Nachsel R, Pötter H, Kuhlmeier D, Carstens C, Gärtner C, Zilch C. Magnetpartikel-basiertes Mikrosystem zum magnetoresistiven Schnellnachweis Sepsis-relevanter Pathogene. Proceedings MikroSystemTechnik Kongress 2012, VDE-Verlag
  • Kuhlmeier D, Gärtig C. Mit der PCR der Parodontitis auf der Spur. BIOspektrum 02.13.
  • Kuhlmeier D, Sandetskaya N, Lorenzen M, Schotter J. Retter im Scheckkartenformat. Laborpraxis 03/2011.
  • Kuhlmeier D, Sandetskaya N, Allelein S. Application of nanotechnology in miniaturized systems and its use in medical and food analysis. Recent Pat Food Nutr Agric. 4 (2012), 3, S. 187-99. DOI dx.doi.org/10.2174/2212798411204030187.
  • Lindner I. et al. alpha 2-Macroglobulin Inhibits the Malignant Properties of Astrocytoma Cells by Impeding beta-Catenin Signaling. Cancer Research, v. 70 (2010), no. 1, p. 277-287.

Patents

  • Kuhlmeier D, Sandetskaya N, Hennig K, Naumann A. Methode zur spezifischen Isolation von Nukleinsäuren. Patent application PCT/EP2013/072331.