Frank F. Bier

Construction of an artificial cell membrane anchor using DARC as a fitting for artificial extracellular functionalities of eukaryotic cells

The need to functionalize cell membranes in a directed way for specific applications as single cell arrays or to force close cell-to-cell contact for artificial intercellular interaction and/or induction concerning stem cell manipulation or in general to have a tool for membrane and cell surface-associated processes, we envisaged a neutral inactive membrane anchor for extracellular entities to facillitate the above mentioned functionalities.The silent Duffy antigen/receptor for chemokines (DARC) is a receptor-like membrane protein of erythrocytes and mediates no cell transduction not at least regarding a missing or truncated G-loop and therefore it seemed to be the candidate for our cell membrane anchor.We isolated the genetic information of DARC from human genomic DNA and cloned it in a mammalian cell line as a fusion protein via a suitable plasmid vector.In this report we demonstrate that the human plasma membrane protein DARC can be used as an artificial anchor molecule in cell surface engineering applications. We constructed the fusion protein SNAP-tag-DARC, consisting of DARC and the self-labeling protein tag SNAP-tag® (Covalys). The SNAP-tag® served as an example for a molecular-technological developed protein that is artificially attached to the extracellular side of the plasma membrane through our DARC-anchor. SnapTag should serve as an example for any extracellular entity and was easy to detect by a commercial detection system. The synthesis of SNAP-tag-DARC, its correct incorporation into the cell membrane and the functionality of the SNAP-tag® were verified by RT-PCR, Western blotting and confocal fluorescence microscopy and showed the desired functionality as an membrane anchor for an extracellular application entity.

Integrated planar optical waveguide interferometer biosensors: a comparative review.

Integrated planar optical waveguide interferometer biosensors are advantageous combinations of evanescent field sensing and optical phase difference measurement methods. By probing the near surface region of a sensor area with the evanescent field, any change of the refractive index of the probed volume induces a phase shift of the guided mode compared to a reference field typically of a mode propagating through the reference arm of the same waveguide structure. The interfering fields of these modes produce an interference signal detected at the sensor׳s output, whose alteration is proportional to the refractive index change. This signal can be recorded, processed and related to e.g. the concentration of an analyte in the solution of interest. Although this sensing principle is relatively simple, studies about integrated planar optical waveguide interferometer biosensors can mostly be found in the literature covering the past twenty years. During these two decades, several members of this sensor family have been introduced, which have remarkably advantageous properties. These entail label-free and non-destructive detection, outstandingly good sensitivity and detection limit, cost-effective and simple production, ability of multiplexing and miniaturization. Furthermore, these properties lead to low reagent consumption, short analysis time and open prospects for point-of-care applications. The present review collects the most relevant developments of the past twenty years categorizing them into two main groups, such as common- and double path waveguide interferometers. In addition, it tries to maintain the historical order as it is possible and it compares the diverse sensor designs in order to reveal not only the development of this field in time, but to contrast the advantages and disadvantages of the different approaches and sensor families, as well.

Determination of p-aminophenol and catecholamines at picomolar concentrations based on recycling enzyme amplification

A bienzyme electrode based on the amplification of a signal has been developed which allows the determination of picoto nanomolar concentrations of p-aminophenol. The active element of the sensor comprised of coimmobilised laccase and glucose dehydrogenase enzymes coupled with an oxygen electrode. Laccase catalyzes p-aminophenol oxidation by oxygen to give p-iminoquinone. The latter is reduced by excess of glucose in the presence of glucose dehydrogenase and results in recycling of the substrate. The detection is realized by measuring the decrease in oxygen concentration. The detection limit for p-aminophenol is 100 pM. The feasibility of the determination of a number of other substrates (polyphenols, polyamines, ferrocene derivatives) in the nanomolar range has been demonstrated. A significant background signal has been found for p-aminophenylphosphate. This background is probably caused by the ability of laccase to catalyze the oxidative dephosphorylation. In the presence of phosphate ions this background is practically completely eliminated. 50 pM of alkaline phosphatase could be determined after a 2 min incubation in p-aminophenylphosphate solution by determination of the p-aminophenol formed as the result of hydrolysis. The whole analysis time does not exceed 5 min. The new technique is suitable for application in alkaline phosphatase based enzyme immunoassays.

Real time analysis of competitive binding using grating coupler immunosensors for pesticide detection

Abstract Real time analysis of binding events is one step forward from immunoassays to immunosensors. Using grating couplers as transducers, the approach is adapted to hapten detection by a competitive assay format. A special advantage of real time analysis is the information obtained from the kinetic behaviour of the immunosystem. In this study we show that the interpretation of the dynamic system response compared to a quasi-stationary one shifts the detection limit to lower hapten concentrations, and shortens the assay time. In the case of s-triazines, it was found that the detection limit decreases by 4 times, while incubation time is shortened tenfold to 3 min.

Real-time measurement of nucleic-acid hybridization using evanescent-wave sensors: steps towards the genosensor

Abstract Nucleic acids are used as receptors in biosensing for the determination of complementary nucleic-acid strands. This may be useful in clinical diagnostics, e.g., searching for DNA from viruses, or in other fields of hygiene or environmental monitoring. In this study we demonstrate the reversible binding of DNA oligonucleotides to immobilized DNA targets. Using both a grating coupler detector and surface plasmon resonance, the evanescent field is employed to distinguish between bound and unbound species. As a bridge for the immobilization of the DNA, streptavidin (or avidin) is coupled to the sensor surface. Three different targets have been investigated: (1) randomly biotinylated poly(dA); (2) 5′-biotinylated 24-mer consisting of balanced amounts of purines and pyrimidines; and (3) 5′-biotinylated 13-mer. The binding kinetics of poly (dT) have been investigated, demonstrating this fast process as multibinding complex formation, since no sequence specificity is involved. The binding of a variety of 13-mers to 24-mer and 13-mer templates has been observed and the binding kinetics analysed. In a 13-mer point mutations can be resolved by analysis of association and dissociation rate constants.

Functional peptide microarrays for specific and sensitive antibody diagnostics

Peptide microarrays displaying biologically active small synthetic peptides in a high‐density format provide an attractive technology to probe complex samples for the presence and/or function of protein analytes. We present a new approach for manufacturing functional peptide microarrays for molecular immune diagnostics. Our method relies on the efficiency of site‐specific solution‐phase coupling of biotinylated synthetic peptides to NeutrAvidin (NA) and localized microdispensing of peptide‐NA‐complexes onto activated glass surfaces. Antibodies are captured in a sandwich manner between surface immobilized peptide probes and fluorescence‐labeled secondary antibodies. Our work includes a total of 54 peptides derived from immunodominant linear epitopes of the T7 phage capsid protein, Herpes simplex virus glycoprotein D, c‐myc protein, and three domains of the Human coronavirus polymerase polyprotein and their cognate mAbs. By using spacer molecules of different type and length for NA‐mediated peptide presentation, we show that the incorporation of a minimum spacer length is imperative for antibody binding, whereas the peptide immobilization direction has only secondary importance for antibody affinity and binding. We further demonstrate that the peptide array is capable of detecting low‐picomolar concentrations of mAbs in buffered solutions and diluted human serum with high specificity.

Fibre-optic genosensor for specific determination of femtomolar DNA oligomers

Abstract The binding of DNA oligonucleotides to immobilized DNA-targets using a fibre optic fluorescence sensor is demonstrated. 13mer oligonucleotides were attached to the core of a multimode fibre. The complementary sequence was detected by use of a fluorescent double strand specific DNA ligand (YOYO and PicoGreen). The evanescent field was employed to distinguish between bound and not bound species. The template DNA-oligomer was immobilized either by direct coupling to the activated sensor surface or using the avidin-biotin bridge. Single base mismatches in the target sequence were detected; and a detection limit of the sensor of 30 fM (3.2 attomoles) was found for the matching target.

Integrated optical immunosensor for s-triazine determination: regeneration, calibration and limitations

Grating couplers, as an example of an integrated optical transducer, were investigated for label-free immunosensing in a flow-through system. The detection of pesticides by a competitive assay format was demonstrated using a triazine (terbutryn) as an example. Long-term stability and reproducibility were achieved by immobilizing the hapten, which in this instance was a derivative of the pesticide to be determined. Hence, the lifetime of the sensor does not depend on the stability of the antibody but is limited only by the quality of the immobilization. Regeneration of the sensor surface was achieved using proteinase supported by acidic washing; it was observed directly and could therefore be optimized. In consequence, calibration, usually critical in immunosensing, was possible. Non-specific binding was determined to be 10% and could be decreased by addition of bovine serum albumin. The detection limit found for terbutryn was 15 µg l–1 in the arrangement used; strategies for enhancement of sensitivity are discussed.

Dielectrophoresis of DNA: Quantification by impedance measurements

Dielectrophoretic properties of DNA have been determined by measuring capacitance changes between planar microelectrodes. DNA sizes ranged from 100 bp to 48 kbp, DNA concentrations from below 0.1 to 70 mugml. Dielectrophoretic spectra exhibited maximum response around 3 kHz and 3 MHz. The strongest response was found for very long DNA (above 10 kbp) and for short 100 bp fragments, which corresponds to the persistence length of DNA. The method allows for an uncomplicated, automatic acquisition of the dielectrophoretic properties of submicroscopical objects without the need for labeling protocols or optical accessibility.

Production of functional antibody fragments in a vesicle-based eukaryotic cell-free translation system.

Cell-free protein synthesis is of increasing interest for the rapid and high-throughput synthesis of many proteins, in particular also antibody fragments. In this study, we present a novel strategy for the production of single chain antibody fragments (scFv) in a eukaryotic in vitro translation system. This strategy comprises the cell-free expression, isolation and label-free interaction analysis of a model antibody fragment synthesized in two differently prepared insect cell lysates. These lysates contain translocationally active microsomal structures derived from the endoplasmic reticulum (ER), allowing for posttranslational modifications of cell-free synthesized proteins. Both types of these insect cell lysates enable the synthesis and translocation of scFv into ER-derived vesicles. However, only the one that has a specifically adapted redox potential yields functional active antibody fragments. We have developed a new methodology for the isolation of functional target proteins based on the translocation of cell-free produced scFv into microsomal structures and subsequent collection of protein-enriched vesicles. Antibody fragments that have been released from these vesicles are shown to be well suited for label-free binding studies. Altogether, these results show the potential of insect cell lysates for the production, purification and selection of antibody fragments in an easy-to-handle and time-saving manner.