Jan Regtmeier

Electrodeless dielectrophoresis for bioanalysis: Theory, devices and applications

Dielectrophoresis is a non‐destructive, label‐free method to manipulate and separate (bio‐) particles and macromolecules. The mechanism is based on the movement of polarizable objects in an inhomogeneous electric field. Here, microfluidic devices are reviewed that generate those inhomogeneous electric fields with insulating posts or constrictions, an approach called electrodeless or insulator‐based dielectrophoresis. Possible advantages compared to electrode‐based designs are a less complex, monolithic fabrication process with low‐cost polymeric substrates and no metal surface deterioration within the area of sample analysis. The electrodeless design has led to novel devices, implementing the functionality directly into the channel geometry and covering many areas of bioanalysis, like manipulation and separation of particles, cells, DNA, and proteins.

Dielectrophoretic Trapping and Polarizability of DNA: The Role of Spatial Conformation

Dielectrophoresis is a convenient tool for controlled manipulation of DNA with numerous applications, including DNA trapping, stretching, and separation. However, the mechanisms behind the dielectrophoretic properties of DNA are still under debate, and the role of conformation has not been addressed yet. Here, we quantify dielectrophoretic effects on DNA by determining its polarizability from microfluidic single molecule trapping experiments. We systematically study different DNA configurations (linear and supercoiled, 6-164 kbp) and demonstrate that the polarizability strongly depends on the specific conformation and size of the DNA molecules. The connection to its spatial extension is established by measuring diffusion coefficients and from that the radii of gyration; details about the spatial DNA structure are obtained from atomic force microscopy images. For linear and supercoiled DNA fragments, we found a power-law scaling for the polarizabilities and the diffusion coefficients. Our results imply a scaling of the polarizability with the radius of gyration, alpha approximately Rg0.9+/-0.1 and alpha approximately Rg1.6+/-0.2 for linear and supercoiled DNA, respectively. As an application, we demonstrate the separation of DNA topoisomers based on their dielectrophoretic properties, achieving baseline resolution within 210 s. Purified DNA samples of specific configuration may be of great importance for DNA nanoassembly or future DNA vaccines.

Physisorbed surface coatings for poly(dimethylsiloxane) and quartz microfluidic devices.

AbstractSurface modifications of microfluidic devices are of essential importance for successful bioanalytical applications. Here, we investigate three different coatings for quartz and poly(dimethylsiloxane) (PDMS) surfaces. We employed a triblock copolymer with trade name F108, poly(l-lysine)-g-poly(ethylene glycol) (PLL-PEG), as well as the hybrid coating n-dodecyl-β-d-maltoside and methyl cellulose (DDM/MC). The impact of these coatings was characterized by measuring the electroosmotic flow (EOF), contact angle, and prevention of protein adsorption. Furthermore, we investigated the influence of static coatings, i.e., the incubation with the coating agent prior to measurements, and dynamic coatings, where the coating agent was present during the measurement. We found that all coatings on PDMS as well as quartz reduced EOF, increased reproducibility of EOF, reduced protein adsorption, and improved the wettability of the surfaces. Among the coating strategies tested, the dynamic coatings with DDM/MC and F108 demonstrated maximal reduction of EOF and protein adsorption and simultaneously best long-term stability concerning EOF. For PLL-PEG, a reversal in the EOF direction was observed. Interestingly, the static surface coating strategy with F108 proved to be as effective to prevent protein adsorption as dynamic coating with this block copolymer. These findings will allow optimized parameter choices for coating strategies on PDMS and quartz microfluidic devices in which control of EOF and reduced biofouling are indispensable. FigureThe difference between static coating (left) and dynamic surface coating (right) in a microfluidic channel is shown schematically. In the static case, the surface is incubated with the coating agent prior to a specific experiment. Dynamic coating refers to the case in which the coating agent is present in the solution during a specific experiment

High resolution imaging of surface patterns of single bacterial cells.

We systematically studied the origin of surface patterns observed on single Sinorhizobium meliloti bacterial cells by comparing the complementary techniques atomic force microscopy (AFM) and scanning electron microscopy (SEM). Conditions ranged from living bacteria in liquid to fixed bacteria in high vacuum. Stepwise, we applied different sample modifications (fixation, drying, metal coating, etc.) and characterized the observed surface patterns. A detailed analysis revealed that the surface structure with wrinkled protrusions in SEM images were not generated de novo but most likely evolved from similar and naturally present structures on the surface of living bacteria. The influence of osmotic stress to the surface structure of living cells was evaluated and also the contribution of exopolysaccharide and lipopolysaccharide (LPS) by imaging two mutant strains of the bacterium under native conditions. AFM images of living bacteria in culture medium exhibited surface structures of the size of single proteins emphasizing the usefulness of AFM for high resolution cell imaging.

Chiral Particle Separation by a Nonchiral Microlattice

We conceived a model experiment for a continuous separation strategy of chiral molecules (enantiomers) without the need of any chiral selector structure or derivatization agents: Microparticles that only differ by their chirality are shown to migrate along different directions when driven by a steady fluid flow through a square lattice of cylindrical posts. In accordance with our numerical predictions, the transport directions of the enantiomers depend very sensitively on the orientation of the lattice relative to the fluid flow.

Particle sorting by a structured microfluidic ratchet device with tunable selectivity: theory and experiment

We theoretically predict and experimentally demonstrate that several different particle species can be separated from each other by means of a ratchet device, consisting of periodically arranged triangular (ratchet) shaped obstacles. We propose an explicit algorithm for suitably tailoring the externally applied, time-dependent voltage protocol so that one or several, arbitrarily selected particle species are forced to migrate oppositely to all the remaining species. As an example we present numerical simulations for a mixture of five species, labelled according to their increasing size, so that species 2 and 4 simultaneously move in one direction and species 1, 3, and 5 in the other. The selection of species to be separated from the others can be changed at any time by simply adapting the voltage protocol. This general theoretical concept to utilize one device for many different sorting tasks is experimentally confirmed for a mixture of three colloidal particle species.

Negative mobility and sorting of colloidal particles

Transport in the realm of soft matter is strongly influenced by diffusion. Conditions far from thermal equilibrium and non-linear dynamics may give rise to unexpected transport phenomena, which are ruled out by the second law of thermodynamics under equilibrium conditions. Here, we highlight one of them, namely the migration of colloidal microparticles opposite to a static force and illustrate its application for particle sorting purposes.

Nanofluidic devices for dielectrophoretic mobility shift assays by soft lithography

We report development and application of 3D structured nano-microfluidic devices that were produced via soft lithography with poly(dimethylsiloxane). The procedure does not rely on hazardous or time-consuming production steps. Here, the nanochannels were created by channel-spanning ridges that reduce the flow height of the microchannel. Several realizations of the ridge layout and nanochannel height are demonstrated, depicting the high potential of this technique. The nanochannels proved to be stable even for width-to-height aspect ratios of 873:1. Additionally, an application of these submicrometer structures is presented with a new technique of a dielectrophoretic mobility shift assay (DEMSA). The DEMSA was used to detect different DNA variants, e.g. protein?DNA-complexes, via a shift in (dielectrophoretically retarded) migration velocities within an array of nanoslits.

Microfluidic carbon-blackened polydimethylsiloxane device with reduced ultra violet background fluorescence for simultaneous two-color ultra violet/visible-laser induced fluorescence detection in single cell analysis

In single cell analysis (SCA), individual cell-specific properties and inhomogeneous cellular responses are being investigated that is not subjected to ensemble-averaging or heterogeneous cell population effects. For proteomic single cell analysis, ultra-sensitive and reproducible separation and detection techniques are essential. Microfluidic devices combined with UV laser induced fluorescence (UV-LIF) detection have been proposed to fulfill these requirements. Here, we report on a novel microfluidic chip fabrication procedure that combines straightforward production of polydimethylsiloxane (PDMS) chips with a reduced UV fluorescence background (83%-reduction) by using PDMS droplets with carbon black pigments (CBP) as additives. The CBP-droplet is placed at the point of detection, whereas the rest of the chip remains transparent, ensuring full optical control of the chip. We systematically studied the relation of the UV background fluorescence at CBP to PDMS ratios (varying from 1:10 to 1:1000) for different UV laser powers. Using a CBP/PDMS ratio of 1:20, detection of a 100 nM tryptophan solution (S/N = 3.5) was possible, providing a theoretical limit of detection of 86 nM (with S/N = 3). Via simultaneous two color UV/VIS-LIF detection, we were able to demonstrate the electrophoretic separation of an analyte mixture of 500 nM tryptophan (UV) and 5 nM fluorescein (VIS) within 30 s. As an application, two color LIF detection was also used for the electrophoretic separation of the protein content from a GFP-labeled single Spodoptera frugiperda (Sf9) insect cell. Thereby just one single peak could be measured in the visible spectral range that could be correlated with one single peak among others in the ultraviolet spectra. This indicates an identification of the labeled protein γ-PKC and envisions a further feasible identification of more than one single protein in the future.

Photolithographic fabrication of arbitrarily shaped SU-8 microparticles without sacrificial release layers

We report on an efficient high throughput method for the photolithographic fabrication of well-defined arbitrarily shaped SU-8 microparticles without a sacrificial release layer. The procedure eliminates the spincoating of a sacrificial layer otherwise needed for particle lift-off, thereby reducing processing time and costs. Statistical analysis of the size distribution revealed a standard deviation of less than 2.3% in size. The particles can be immediately released into aqueous solution. This allows for anisotropical functionalization of the particles with, for example, biological loads or elements of molecular recognition after the development of the SU-8 structures.