Projects

Biocompatible materials and surfaces

Biocompatible materials and surfaces are needed for the development of bio-analytical micro devices. Compared with the macro scale laboratory equipment, these micro devices have a higher surface to volume ratio (SVR) accordant to their design. In the most cases this unfa-vorable high SVR restricts the outcome of a wide range of revolutionary applications, e.g. in the point-of-care diagnostics.

Many of these micro devices are made of polymer materials whose surface effects can dis-rupt the often delicate and optimized concentrations of composite reaction mixtures. Possible surface effects are direct interactions with and passive adsorption of chemical compounds.

We are searching for new polymer materials which can be used for a wide range of biocom-patible applications like enzymatic reactions, extraction and purification methods and detec-tion systems. We also investigate biocompatible surface treatments and coatings in order to overcome inhibitory surface effects. The implementation of surface related reaction mixture additives are also promising to enhanced biochemical reactions like shown in Figure 1, illustrating PCR amplification efficiency in dependence to the concentration of PVP (Polyvinyl-pyrrolidone) as additive.

Fig. 1: Influence of different Polyvinylpyrrolidone (PVP) concentrations on standardized quantitative PCR measurements in the LightCycler® 1.5 (Roche) with 20 µl glass capillaries.

Biochip technology platforms for microarray applications

We develop microarray technology platforms for DNA biochip analysis (for example subtyp-ing of human papilloma virus [HPV]), protein analysis (competitive assays for different clinical parameters like microglobulin-2β, HbA1C or cytokines), on-chip-PCR and on-chip-NASBA (Nucleic Acid Sequence-Based Amplification) analyzing genomic DNA and gene expression simultaneously.

A new 3D surface chemistry improving microarray technology

Since the first presentation in 1995 microarrays have been a growing field of interest, mainly because of their enormous potential in genomic and proteomic research.

However, one chip is typically used for a single analytical problem and then disposed. Be-sides, one of the key problems in the development of biochips for a given analytical problem is the determination of suitable chip parameters such as probe affinities and optimized hy-bridization conditions like buffer concentrations and temperature. Major drawbacks are lack of robustness, loss of sensitivity and specificity compared to conventional methods, the con-sumable costs and costs for the development time preventing their use in routine application and diagnostics.

Therefore, we developed a novel three dimensional surface chemistry consisting of a water soluble polymer. With this polymer probes for protein or DNA analysis can be immobilized on matrices such as plastic or glass by using UV photo cross-linkage (254 / 365nm).

Fig. 2: This model illustrates the differences between a two dimensional (2D) and a three dimensional (3D) surface. The immobilization of the probes takes place with a 5´-modification (15 mer thymine-tail). In the three dimensional manner the probes can be immobi-lized on the surface of the substrate and the polymer network. The polymer network itself consists of three statistically disposed components (DMAA, MABP and VPA). The labelled PCR amplicon (green indicated) hybridizes on the immobilized probe. PMMA can also be used as well as glass substrates.

Bioanalytical cryogels

Cryogels are porous hydrogels prepared from monomeric and/or polymeric precursor solutions which are polymerized and/or crosslinked in the frozen state. Typically, aqueous precursor solutions are used which phase separate upon freezing into ice crystals and liquid microphases containing the concentrated precursors and unfrozen water. The initiated gelation reactions are then restricted to the liquid microphases, while the ice acts as a porogen. After gel formation is completed, the system is thawed to obtain a solid with interconnected pores.

In our group, we have developed a highly efficient and versatile method for the preparation of biofunctional, microstructured and surface-attached cryogels within a single reaction step (DOI: 10.1021/acsami.7b01232). To this end, we apply an aqueous solution containing a photo-reactive copolymer as the single gel forming precursor and the desired biomolecules to be immobilized in the gels. After freezing, this mixture is briefly illuminated with UV-light to induce non-specific C,H-insertion reactions leading to a simultaneous crosslinking of the polymer chains, covalent immobilization of the contained biomolecules and surface-attachment of the forming gels to almost any kind of polymeric substrate. Further, the gels can easily be microstructured by using a photomask during the UV-exposure, as known from classical photolithography (Figure 1). Using this process we study the preparation of functional cryogel elements containing various biomolecules like DNA or proteins and their integration into different analytical platforms. An example of such a platform developed in our group for an application in the point-of-care diagnostics (DOI: 10.1021/acs.analchem.7b01182) is shown in Figure 2.

Figure 1. Top: Schematic depiction of the developed method for the preparation of biofunctional, microstructured and surface-attached cryogels in a single reaction step. Bottom: Micrographs of fabricated cryogels (source: DOI: 10.1021/acsami.7b01232).

Figure 2. Assembly of drawings, white light and fluorescence images to illustrate the preparation and application of a cryogel based analytical platform. The device consists of a linear assembly of individually functionalized cryogel monoliths directly prepared and anchored in a transparent glass capillary with an inner diameter of 450 µm. In the shown example antibodies against the inflammatory cytokine IL-6 were immobilized in the central compartment. The other two compartments were modified in order to serve as negative (left) and positive (right) controls. The samples to be analyzed were transferred through the device followed by a short staining step using a fluorescence labeled secondary antibody. Typical results of a positive and negative test are shown. The achieved limit of detection (LOD) for IL-6 in undiluted human blood plasma was 30 pg/ml (source: DOI: 10.1021/acs.analchem.7b01182).

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