Thomas Schalkhammer

Capillary electrophoresis with on‐chip four‐electrode capacitively coupled conductivity detection for application in bioanalysis

Microchip capillary electrophoresis (CE) with integrated four‐electrode capacitively coupled conductivity detection is presented. Conductivity detection is a universal detection technique that is relatively independent on the detection pathlength and, especially important for chip‐based analysis, is compatible with miniaturization and on‐chip integration. The glass microchip structure consists of a 6 cm etched channel (20 νm×70 νm cross section) with silicon nitride covered walls. In the channel, a 30 nm thick silicon carbide layer covers the electrodes to enable capacitive coupling with the liquid inside the channel as well as to prevent interference of the applied separation field. The detector response was found to be linear over the concentration range from 20 νM up to 2 mM. Detection limits were at the low νM level. Separation of two short peptides with a pI of respectively 5.38 and 4.87 at the 1 mM level demonstrates the applicability for biochemical analysis. At a relatively low separation field strength (50 V/cm) plate numbers in the order of 3500 were achieved. Results obtained with the microdevice compared well with those obtained in a bench scale CE instrument using UV detection under similar conditions.

High-throughput assays on the chip based on metal nano-cluster resonance transducers

High throughput transducers using metal cluster resonance technology are based on surface-enhancement of metal cluster light absorption. These devices can be used for detection of biorecognitive binding, as well as structural changes of nucleic acids, proteins or any other polymer. The optical property for the analytical application of metal cluster films is the so-called anomalous absorption. An absorbing film of clusters positioned 10--400 nm to a mirror surface reacts in a similar way to a reflection filter. At a certain distance of the absorbing layer to the mirror the reflected electromagnetic field has the same phase at the position of the absorbing cluster as the incident fields. This feedback mechanism strongly enhances the effective cluster absorption coefficient. The system is characterised by a narrow reflection minimum whose spectral position shifts sensitively with the interlayer thickness, because a given cluster-mirror distance and wavelength defines the optimum phase. Based on this principle a set of novel tools including biochips and micro arrays is presented, which enabled us to transduce binding, as well as changes of protein-, DNA- and polymer-conformation, quantitatively into an optical signal which can be observed directly as a colour change of a sensor-chip surface.

Surface Enhanced Resonance of Metal Nano Clusters: A Novel Tool for Proteomics

A novel ‘nano’-tool based on metal clusters enables the transduction of changes of biorecognitive binding as well as conformation quantitatively into an optical signal which can be observed directly as a color change of a sensor-chip surface.Proteins including various enzymes and serum proteins were spotted via micro-arraying onto the chip surface forming monolayer and thin film dots. Multi-layered (up to 300 nm thick) nano-gel-pads were stabilized by photo crosslinking the protein dots with UV-light. By deposition of metal-nano clusters, synthesized via chemical means or sputter coating on top of photo-crosslinked macromolecules an optical resonance effect was obtained.The response of the novel surface enhanced absorption cluster sensor-chip was transduced spectroscopically in the visible and IR range of the spectrum via an 8 μm resolution reflectance scanner.

Food-allergen assays on chip based on metal nano-cluster resonance

A new nano technique based on enhanced metal cluster absorption was used to detect antibody-food allergen interactions, resulting in a optical signal, which is observed directly as a color change of the chip surface either with the eye or using an optical scanner.

Design and Peptide-Based Validation of Phage Display Antibodies for Proteomic Biochips

To validate potential application of phage display-antibody arrays for high-throughput screening on a novel proteomics biochip, we examined the epitopes versus the full protein of glucose-6-phosphate-dehydrogenase (G6PD) from yeast. In a predictive approach, we used the Hopp-Woods method and compared the results with antibodies directed against the entire enzyme. In total, 16 peptides of a length of 11 amino acids each fulfilling the desired criteria were identified and synthesized. Subsequently, antibodies against G6PD were raised using a phage display library. Selective interaction of the antibodies with certain peptides facilitated the identification of epitopes predicted by the hydropathic profile. The setup was adapted to a novel biochip system based on surface-enhanced absorption for direct CCD-camera based screening.

DNA biochips based on surface-enhanced fluorescence (SEF) for high-throughput interaction studies

Molecular interaction especially biorecognitive binding can be visualized by metal cluster enhanced fluorescence. Fluorescent molecules that are bound within the electromagnetic field of a layer of metal clusters exhibit a strong boost in excitation as well as emission. We present a study, using novel surface enhanced chips in glass-slide-format, their set up, the micro arraying onto the surface, and the hybridization of oligonucleotides on these chips. Compared to standard (glass slide) DNA chips, performance, fluorescent signals as well as signal to noise ratio were considerably higher.