Advanced Analytical Methods for Pharmaceutical and Diagnostic Applications

Driving forces for the development of novel analytical technologies in the life-science industry are described. Technologies which either were developed in Bio-Analytical Research or brought to a reliability required for routine applications will be elucidated and, on the basis of practical examples, the impact of modern analytical technologies on the industrial research and development will be discussed: Optical biosensors based on evanescent excitation of luminescence allow for real-time monitoring of the binding of active compounds to specific biomolecular recognition sites. Molecular imaging technologies have the potential to gain rapid access to physical maps of genomic materials. Capillary electrophoresis or affinity gel electrophoresis are well suited for the fast determination of oligonucleotide mixtures in nl amounts of samples. Integrated capillary electrophoresis on chips will allow to multiplex capillary systems at low costs and results in high separation efficiencies. MALDI-TOF MS is an easy to operate non-scanning mass spectrometric instrumentation for the analysis of high molecular weight biopolymers such as immunoglobulins

Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence

Grell E, Pawlak M, Anselmetti D, Schick E, Lewitzki E, Ehrat M. Na,K-ATPase on a waveguide sensor : supramolecular assembly and side directed binding studies by surface-confined fluorescence. In: Taniguchi K, Kaya S, eds. Na/K-ATPase and related ATPases: proceedings of the 9th International Conference on the Na/K-ATPase and Related ATPases. Excerpta Medica international congress series. Vol 1207. Amsterdam: Elsevier; 2000: 437-440.The functional assembly of FITC-Na,K-ATPase membrane fragments on a surface-modified Ta2O5 waveguide allows to investigate the directed binding of ligands by surface-confined fluorescence studies. The results allow to draw conclusions about the sidedness of interactions. The fluorescence intensity decrease observed upon the selective binding of K+ is attributed to its coordination to a site accessible from the former intracellular membrane side

DNA and Protein Microarrays and their Contributions to Proteomics and Genomics

Knowledge in genomics and proteomics has exploded in the last two decades. This is in part due to key developments that have revolutionized the possibilities of bioanalytics such as the introduction of polymerase chain reaction (PCR) in the mid 80s that formed the base for the massively parallel sequencing of the genomes.A few years ago DNA and protein microarray analysis were added to the toolbox of life sciences analytics. These technologies already proved to be ideal tools for the identification of gene targets, the simultaneous measurement of the expression of a high number of genes or proteins, and the increase of the level of understanding of the biological functions of genes and proteins. A small number of experiments are now sufficient to obtain information on gene or protein expression which could not be obtained by using conventional bioanalytical technologies or which required an extremely high experimental effort. In the future applications, high sensitivity DNA and protein microarrays will allow low abundant genes and proteins to be monitored that so far have been inaccessible to current microarray technologies and thus will generate a new dimension of genomic and proteomic information

Protein Microarrays Based on Polymer Brushes Prepared via Surface-Initiated Atom Transfer Radical Polymerization

Polymer brushes represent an interesting platform for the development of high-capacity protein binding surfaces. Whereas the protein binding properties of polymer brushes have been investigated before, this manuscript evaluates the feasibility of poly(glycidyl methacrylate) (PGMA) and PGMA-co-poly(2-(diethylamino)ethyl methacrylate) (PGMA-co-PDEAEMA) (co)polymer brushes grown via surface-initiated atom transfer radical polymerization (SI-ATRP) as protein reactive substrates in a commercially available microarray system using tantalum-pentoxide-coated optical waveguide-based chips. The performance of the polymer-brush-based protein microarray chips is assessed using commercially available dodecylphosphate (DDP)-modified chips as the benchmark. In contrast to the 2D planar, DDP-coated chips, the polymer-brush-covered chips represent a 3D sampling volume. This was reflected in the results of protein immobilization studies, which indicated that the polymer-brush-based coatings had a higher protein binding capacity as compared to the reference substrates. The protein binding capacity of the polymer-brush-based coatings was found to increase with increasing brush thickness and could also be enhanced by copolymerization of 2-(diethylamino)ethyl methacrylate (DEAEMA), which catalyzes epoxide ring-opening of the glycidyl methacrylate (GMA) units. The performance of the polymer-brush-based microarray chips was evaluated in two proof-of-concept microarray experiments, which involved the detection of biotin-streptavidin binding as well as a model TNF alpha reverse assay. These experiments revealed that the use of polymer-brush-modified microarray chips resulted not only in the highest absolute fluorescence readouts, reflecting the 3D nature and enhanced sampling volume provided by the brush coating, but also in significantly enhanced signal-to-noise ratios. These characteristics make the proposed polymer brushes an attractive alternative to commercially available, 2D microarray surface coatings

Analysis of Drug/Plasma Protein Interactions by Means of Asymmetrical Flow Field-Flow Fractionation

Purpose. The applicability of Asymmetrical Flow Field-Flow Fractionation (Asymmetrical Flow FFF) as an alternative tool to examine the distribution of a lipophilic drug (N-Benzoyl-staurosporine) within human plasma protein fractions was investigated with respect to high separation speed and loss of material on surfaces due to adsorption. Methods. Field-Flow Fractionation is defined as a group of pseudo-chromatographic separation methods, where compounds are separated under the influence of an externally applied force based on differences in their physicochemical properties. This method was used to separate human plasma in its protein fractions. The drug distribution in the fractions was investigated by monitoring the fractionated eluate for drug content by fluorescence spectroscopy. Results. Human plasma was separated into human serum albumin (HSA), high density lipoprotein (HDL), α2-macroglobulin and low density lipoprotein (LDL) fractions in less than ten minutes. Calibration of the system and identification of the individual fractions was performed using commercially available protein reference standards. The influence of membrane type and carrier solution composition on the absolute recovery of N-Benzoyl-staurosporine and fluorescein-isothio-cyanate-albumin (FITC-albumin) was found to be quite significant. Both factors were optimized during the course of the investigations. N-Benzoyl-staurosporine was found to be enriched in the fraction containing HSA. Conclusions. If experimental conditions are thoroughly selected and controlled to suppress drug and plasma protein adsorption at the separation membrane, Asymmetrical Flow FFF shows high recoveries and fast separation of human plasma proteins, and can be a reliable tool to characterize drug / plasma protein interactions. For analytical purposes it has the potential to rival established technologies like ultracentrifugation in terms of ease-of-use, precision, and separation tim

Fluorescent vesicles for signal amplification in reverse phase protein microarray assays

Developments in microarray technology promise to lead to great advancements in the biomedical and biological field. However, implementation of these analytical tools often relies on signal amplification strategies that are essential to reach the sensitivity levels required for a variety of biological applications. This is true especially for reverse phase arrays where a complex biological sample is directly immobilized on the chip. We present a simple and generic method for signal amplification based on the use of antibody-tagged fluorescent vesicles as labels for signal generation. To assess the gain in assay sensitivity, we performed a model assay for the detection of rabbit immunoglobulin G (IgG) and compared the limit of detection (LOD) of the vesicle assay with the LOD of a conventional assay performed with fluorescent reporter molecules. We evaluated the improvements for two fluorescence-based transduction setups: a high-sensitivity microarray reader (ZeptoREADER) and a conventional confocal scanner. In all cases, our strategy led to an increase in sensitivity. However, gain in sensitivity widely depended on the type of illumination; whereas an approximately 2-fold increase in sensitivity was observed for readout based on evanescent field illumination, the contribution was as high as more than 200-fold for confocal scanning