Research

Droplet based mechanical manipulations and the development of electrowetting on dielectrics (EWOD)-microdevices for biological samples including DNA, proteins, and whole cells are under intense investigation and reveal promising applications. These digital microfluidic devices offer multiple possibilities as a lab-on-chip platform for preparation, manipulation, and analysis. For microfluidic droplet transport an open EWOD module was built up. This module is intended to transport bacteria suspensions in droplets, merge with other droplets like dye solutions and separate droplets e.g. for washing processes. Therefore an electrode array composed of 2 times 8 indium tin oxide (ITO) electrodes on glass substrates was structured (Fig. 1).

The array was coated with a hydrophobic material to archive a high contact angle to water droplets. By applying a voltage on the active electrode, electrowetting took place – the contact angle shrunk. Different materials (SU8 photoresist, Cytop, and Parylene) were investigated to archive the best initial contact angle and electrowetting effect (Fig. 2). With a sequential switching of the applied voltage to the neighboring electrodes we were able to transport a droplet of 5 µl on the array (Fig. 3).

Successfully, a modified extended-gate field-effect transistor (EGFET) was developed to detect bacteria. The EGFET is composed of a transducer (MOSFET/OFET) and a sensor area which was functionalized by 5,15-A₂BC-type porphyrins with two specific ligands on the meso-position (Figure 1). One ligand connects the porphyrin ring with a gold surface through a thioacetyl group. The second ligand is characterized by a specific peptide chain which can be connected to the outer membrane of Gram-negative bacteria. With fluorescence liftime imaging microcopy (FLIM) it could clearly be shown that bacteria are immobilized on a gold electrode functionalized with the 5,15-A₂BC-type porphyrin (Figure 2). Further, more negative current values were recorded with the EGFET if bacteria are attached to the electrode's surface (Figure 3)

Electrical microbial characterization is emerging as a key tool of increasing interest for the quantification of microbial properties in the field of microbial communities, community assessment, medical applications, industrial bioprocesses, and the control of environmental systems. Here, oscillating currents in suspensions of different Gram-positive and -negative bacteria were found obtained by applying a direct current (DC)-voltage. To understand the origin of these effects, all measurements (>250) are currently analyzed using an automated R-script. In parallel the measuring chamber was scaled up to 70 ml. This now allows for analyses of a suitable number of bacteria for transcriptome (mRNA) analyses to understand the bacterial answer of the applied current in comparison to unstimulated bacteria.

Dielectric particles in liquids, such as biological cells in a medium, are subjected to a force in a non-uniform alternating electric field depending on their dielectric properties. The phenomenon is called dielectrophoresis (DEP) in contrast to the exposure of charged particles to a uniform constant electric field, named electrophoresis. By applying a dielectrophoretic force onto the bacteria, it is possible to accelerate them in a targeted direction and thus concentrate them in specific regions of an electrode geometry. Ongoing investigations have already confirmed that a large area coverage of the electrodes with bacteria (Fig. 1) can be achieved with a negative dielectrophoretic force. This demonstrated to be possible for different sample fluids and bacterial strains. With the help of statistical experimental design and supporting simulation (Fig. 2), further optimization of the process is now being carried out. Dielectrophoresis is also a method that makes it possible to separate bacteria from each other based on very small physiological differences. This method is intended to be used for sorting bacteria. Appropriate results will flow into the current research topics.

Escherichia coli bacteria during dielectrophoresis over indium tin oxide electrodes

Movie 1 (3x replay speed): At the beginning, bacteria were concentrated at the edges of the electrodes by applying a positive dielectrophoretic force. In this process, they aligned themselves along the electric field lines due to polarization. Increasing the frequency of the electrical voltage from the kilohertz to the megahertz range causes the force to decrease to a value of zero, this can be seen by the bacteria detaching from the edges. A further increase of the frequency results in a reversal of the sign, so that a negative force accelerates the bacteria onto the electrodes. Movie 2 (4x replay speed): The bacteria are accelerated during negative dielectrophoresis in a wide area above the electrodes along the electric field lines from the high potential to the low potential of the electrodes.

Bacteria like various Geobacter or Shewanella strains are known to naturally reduce iron. The organisms use this biochemical strategy to replace oxygen as terminal acceptor in the energy (ATP) generating electron transport chain utilizing an anaerobic iron respiration. This process performed on electrodes can lead to the generation of electrical power.

To identify and obtain a useful combination of bacteria converting glycerol into electrical power, we used wastewater from a local wastewater treatment plant and enriched desired bacteria using a special incubator combined with a potentiostat by growing them in the presence of electrical power with glycerol as carbon source. Beside others, two bacterial species were found to be present in the glycerol cultivation, G. sulfurreducens and R. electrica. Based on these results, further experiments were performed with mixed cultivations of both strains. Glycerol
conversion assays showed, that R. electrica was converting glycerol into acetate which in turn served as carbon source for G. sulfurreducens to generate electrical power at the anode. Developing to one of our new key players, we solved the genome sequence of R. electrica and deduced a pathway for glycerol metabolization. The genome sequence and the ability of acetate consumption by G. sulfurreducens were known before. However, the glycerol utilization assays showed, that R. electrica is excreting large amounts of acetate during glycerol utilization, however, is re-importing and consuming acetate when the glycerol is used up. Moreover, byproducts including formate and ethanol were formed.

In this project, the two bacteria consortia (G. sulfurreducens and R. electrica) will be supplemented with bacteria (defined and mixed) utilizing other cheap bulk/waste products (e.g. cellulose, waste-oils, lignin, aromatic hydrocarbons, and others). Bacteria will be identified using the experimental arrangement used for the isolation of bioelectrically active bacteria outlined above.

Spectroscopic ellipsometry analyzes the changes in polarization state of light after reflection or transmission from a surface and determines the optical properties and thickness of the film. These changes in polarization states are studied by the amplitude ratio (Ψ) and the phase difference (Δ) between p- and s-polarized light, respectively.  Conventional spectroscopic ellipsometry is limited to monitor bacteria due to the low refractive index contrast between bacterium and its ambient. For this reason, total internal reflection ellipsometry (TIRE) consist of Kretschmann configuration is used to monitor bacterial binding to the functionalized layer.

In this configuration surface plasmons (SPs) are coupled with an incoming light beam and excited at a specific incidence angle at the metal-dielectric interface. This matching condition causes surface plasmon resonance (SPR) as a sharp dip in reflected light. SPR detects the changes in the refractive index of the sample environment which is in contact to the metal surface. This measurement technique is real-time and non-destructive which makes it reliable for precise detection of bacteria.

The aim of this research is to detect Gram-negative bacterium Escherichia coli K12 (E. coli K12), a common well investigated apathogenic model strain, using TIRE structure. The measurements are performed by a commercial ellipsometry (SENTECH SE850 ) at Hanover Center for Optical Technologies (Fig. 1). Here, the ellipsometric parameters (Δ and Ψ) are measured and analyzed before and after bacterium injection (Fig. 2). The schematic of the setup is shown in Figure 3.